Proteopedia - User contributions [en]

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:48:50Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/2'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Dimerisation_helices/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the The β2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Recognition_helices/1'> α3 </scene> of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:47:23Z

Meng Han Liu: /* Crystal structure of RTP */

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:46:31Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/2'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Dimerisation_helices/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Recognition_helices/1'> α3 </scene> of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:40:29Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/2'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Recognition_helices/1'> α3 </scene> of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:40:03Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/2'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Recognition_helices/1'> α4 </scene> of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:39:54Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/2'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:31:19Z

Meng Han Liu: /* In ''E. coli'': Tus-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/2'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:28:42Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'> α4 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:26:08Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'> recognition α3 helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:24:45Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Heterodimerisation_domain/1'>recognition (α3) helices </scene> of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T02:15:02Z

Meng Han Liu: /* In ''E. coli'': Tus-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Lock_complex/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T01:59:33Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus-ter_lock/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T01:58:23Z

Meng Han Liu: /* Crystal structure of Tus when bound to ''Ter'' */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as [[Secondary structure|α+β structures]], made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus-ter_lock/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-23T01:47:36Z

Meng Han Liu: /* Crystal structure of Tus when bound to ''Ter'' */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal domain</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal domain</scene>. Both domains are classified as α+β structures, made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. ''Ter'' binding motif occurs in the interdomain <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>β cleft</scene> of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus-ter_lock/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T17:56:17Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal</scene> domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>interdomain</scene> β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus-ter_lock/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Rtp_opening/1' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T17:44:19Z

Meng Han Liu: /* Mechanism of polar fork arrest */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal</scene> domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>interdomain</scene> β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x600px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus-ter_lock/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T17:41:49Z

Meng Han Liu: /* In ''E. coli'': Tus-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/N_terminal/1'>N-terminal</scene> and a smaller <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/C-terminal/2'>C-terminal</scene> domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Interdomain/1'>interdomain</scene> β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/1'>non-permissive side</scene>. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Non-permissive_side/2'>permissive side</scene>. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/L1_loop/1'>L1 loop</scene> of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a <scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus-ter_lock/1'>locked complex</scene> <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T14:50:42Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus_open_scene/1'/>

<scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus_open_scene/1'>TextToBeDisplayed</scene>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T14:32:01Z

Meng Han Liu: /* In ''E. coli'': Tus-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus_main_scene/1'/>

<scene name='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus_main_scene/1'>TextToBeDisplayed</scene>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T14:10:16Z

Meng Han Liu: /* In ''E. coli'': Tus-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_/Tus_-_initial_scene/1'/>

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T12:45:15Z

Meng Han Liu: /* RTP-replisome interaction */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam ''et al'' showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T12:42:24Z

Meng Han Liu: /* Crystal structure of Tus when bound to ''Ter'' */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T10:20:48Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The ''Ter'' sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the ''Ter'' sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity for the RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere ''et al'' suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric ''TerI'' B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian ''et al'' showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to ''Ter'' site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to ''Ter'' site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-''Ter'' complex at check point ''Ter'' site was able to arrest replication fork ''in vivo'' <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin ''et al'' demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T10:00:21Z

Meng Han Liu: /* Summary */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-''Ter'' and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T09:58:32Z

Meng Han Liu: /* Introduction */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and fork fusion. This requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

===Biological roles of replication fork traps===

As a matter of fact, it is not entirely essential to have replication fork traps in ''E. coli'' and ''B. subtilis'' to terminate DNA replication. This is because no deleterious effects were observed with respect to ''tus'' and ''rtp'' gene deletion experiments <ref name="Duggin 2005" />. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-Ter and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T08:52:46Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing terminator (''Ter'') sites, terminator protein and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Main secondary structures
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-Ter and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:27:59Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone [http://en.wikipedia.org/wiki/Cysteine ''Cysteine''] residue is replaced by [http://en.wikipedia.org/wiki/Serine ''Serine''] and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |30
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-replisome interaction|
|-
| Mechanism of fork arrest
| align="center" |
*primary: interaction model
*back-up: clamp model
| align="center" |both RTP-Ter and RTP-replisome contacts involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:12:39Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:08:53Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:08:20Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:04:56Z

Meng Han Liu: /* Crystal structure of RTP */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.

RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:02:48Z

Meng Han Liu: /* ''Ter'' sites */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites is not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:01:53Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction). Adapted from <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T05:01:06Z

Meng Han Liu: /* ''Ter'' sites */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T04:59:31Z

Meng Han Liu: /* In ''B. subtilis'': RTP-''Ter'' complex */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites. Adapted from <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-22T04:43:02Z

Meng Han Liu: /* Introduction */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria use a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T16:34:54Z

Meng Han Liu: /* Summary */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T16:33:28Z

Meng Han Liu: /* Summary */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E. coli'' and ''B. subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

{| class="wikitable"
|+Comparing termination (''Ter'') sites, terminator protein features and replication fork arrest mechanism in ''E. coli'' and ''B. subtilis''.
!
! scope="col" |''E. coli''
! scope="col" |''B. subtilis''
|-
| Number of ''Ter'' sites
| align="center" |10
| align="center" |9
|-
| Size of each ''Ter'' site (bp)
| align="center" |23
| align="center" |16
|-
| Symmetry of ''Ter'' sites
| align="center" |asymmetric
| align="center" |peudo-symmetric
(A and B sites)
|-
| Name of the terminator protein
| align="center" |Termination utilisation substance (Tus)
| align="center" |Replication terminator protein (RTP)
|-
| Size of this protein (kDa)
| align="center" |36 (monomer)
| align="center" |29 (dimer)
|-
| Protein description
| align="center" |asymmetric helix clamp
| align="center" |winged-helix
|-
| Protein topology
| align="center" |α+β
| align="center" |α+β
|-
| Stoichiometry of protein-''Ter'' binding
| align="center" |one monomer binds each ''Ter'' site
| align="center" |two dimers bind each ''Ter'' site
|-
| Key ''Ter'' binding motif
| align="center" |interdomain cleft
| align="center" |recognition helix (α3)
|-
| Groove of DNA insertion
| align="center" |major
| align="center" |major and minor
|-
| Basis of polarity in fork arrest
| align="center" |
*asymmetric interaction between Tus and ''Ter''
*asymmetric structure of Tus
| align="center" |
*differential binding affinity to A and B site
*asymmetric RTP-''Ter'' complex
|-
| Mechanism of fork arrest
| align="center" |
*primary: helicase-specific model
*back-up: locked-complex model
| align="center" |both helicase-specific and locked-complex models involved
|}

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T15:28:27Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

[[Image:Mechanism for polar fork arrest by Tus.jpg|thumb|right|600x330px|alt=polar fork arrest mechanism|How polar replication fork arrest may be achieved by the Tus-''Ter'' complex. Extracted from <ref name="Kaplan 2009" />.]]
====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

File:Mechanism for polar fork arrest by Tus.jpg

2011-05-21T15:05:45Z

Meng Han Liu:

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T14:55:41Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-''Ter'' arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

[[Image:B.Subtilis consensus Termination sequence.png|thumb|right|400x100px|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png||thumb|right|400x325px|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]
===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

<Structure load='1F4K' size='400' frame='true' align='left' caption='The RTP-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T14:26:31Z

Meng Han Liu: /* The "Interaction" model */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model encapsulates the idea that since [http://en.wikipedia.org/wiki/DnaB_helicase helicase DnaB] is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in ''E. coli'' such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.png|frame|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png|frame|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T14:12:02Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model encapsulates the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand during , protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies demonstrates this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in ''E. coli'' is to employ both models as a “fail-safe mechanism” <ref name="Kaplan 2009" />. As the replication machinery approaches the Tus-''Ter'' complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.

On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier <ref name="Wake 1997" /> as well as strand separation by DnaB causes progressive loss of Tus-''Ter'' contacts <ref name="Muclair 2006" />. As a result Tus can easily dissociate and the replisome passes through the ''Ter'' site.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 ''Ter'' sites (I-IX) are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.png|frame|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png|frame|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T08:43:43Z

Meng Han Liu:

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices are biased and mainly face towards the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model encapsulates the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand during , protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies demonstrates this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 Ter sites (I-IX)are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.png|frame|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png|frame|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006"> G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001" />.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T06:57:22Z

Meng Han Liu: /* ''Ter'' sites */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model proposes the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand during , protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies demonstrates this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 Ter sites (I-IX)are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.png|frame|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]
[[Image:Helicase approaching from B site.png|frame|alt=replication fork|Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006">G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001">.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T06:55:00Z

Meng Han Liu: /* ''Ter'' sites */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model proposes the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand during , protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies demonstrates this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 Ter sites (I-IX)are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.png|frame|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006">G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001">.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T06:53:26Z

Meng Han Liu: /* ''Ter'' sites */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model proposes the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand during , protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies demonstrates this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 Ter sites (I-IX)are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.png|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006">G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001">.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

File:B.Subtilis consensus Termination sequence.png

2011-05-21T06:52:48Z

Meng Han Liu: uploaded a new version of "Image:B.Subtilis consensus Termination sequence.png"

B.subtilis concensus termination sequence showing A and B sites

User:Meng Han Liu/DNA termination of replication in E. coli & B. subtilis

2011-05-21T06:51:59Z

Meng Han Liu: /* ''Ter'' sites */

==Introduction==

In prokaryotes such as [http://en.wikipedia.org/wiki/E.coli ''E. coli''] and [http://en.wikipedia.org/wiki/B._subtilis ''B. subtilis''], chromosomal DNA exists in a circular fashion whereby [[DNA Replication, Transcription and Translation#DNA replication|DNA replication]] takes place at a common [http://en.wikipedia.org/wiki/Origin_of_replication#Prokaryotic origin] (''oriC'') <ref name="Duggin 2009"> Duggin, I.G. and S.D. Bell, Termination Structures in the Escherichia coli Chromosome Replication Fork Trap. Journal of Molecular Biology, 2009. 387(3): p. 532-539</ref>. Two [http://en.wikipedia.org/wiki/Replication_fork replication forks] move bidirectionally from ''oriC'' to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes <ref name="Wake 1997"> Wake, R.G. and G.F. King, A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms. Structure, 1997. 5: p. 1-5</ref>. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of ''oriC'' <ref name="Duggin 2008"> Duggin, I.G., Wake, R. Gerry, Bell, Stephen D. Bell and Hill, Thomas M., The replication fork trap and termination of chromosome replication. Molecular Microbiology, 2008. 70(6): p. 1323-1333</ref>. Bacteria uses a [http://en.wikipedia.org/wiki/Circular_bacterial_chromosome#Termination “replication fork trap”] system for successful termination of replication and this requires two factors:
#DNA terminator (''Ter'') sites
#A specific terminator protein that can bind ''Ter''

[[Image:Circular chromosomes of E. coli & B. subtilis.jpg|thumb|500x275px|alt=Circular chromosomes of E. coli and B. subtilis|The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (''Ori C''), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from <ref name="Duggin 2005"> Duggin, I.G. and J.A. Wilce, Termination of replication in bacteria. eLS2005: John Wiley & Sons, Ltd</ref>.]]

===DNA terminator (''Ter'') sites===

''Ter'' is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) <ref name="Kamada 1996"> Kamada, K., et al., Structure of a replication-terminator protein complexed with DNA. Nature (London), 1996. 383(6601): p. 598-603</ref>. In both ''E. coli'' and ''B. subtilis'', multiple ''Ter'' sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, ''Ter'' sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork <ref name="Duggin 2005" />. A suggestive reason for the presence of multiple ''Ter'' sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost ''Ter'' sites.

===DNA terminator proteins===

DNA terminator proteins are proteins that can recognize and bind ''Ter'' DNA to form a complex in order to achieve polar trapping of replication forks <ref name="Duggin 2005" />. In ''E. coli'', this protein is called Tus (terminus utilization substance) whilst in ''B. sutilis'', it is called RTP (replication termination protein). Based on experimental data using mutated ''Ter'' sites and DNA terminator mutants suggests that both protein-DNA (terminator-''Ter'') and protein-protein (terminator-[http://en.wikipedia.org/wiki/Replisome replisome]) interactions are important for successful polar fork arrest <ref name="Kaplan 2009"> Kaplan, D.L.a.B., D., Mechanisms of polar arrest of a replication fork. Molecular Microbiology, 2009. 72(2): p. 279-285</ref>.

==In ''E. coli'': Tus-''Ter'' complex==
[[Image:Ter sites in E. coli.jpg|thumb|left|300x200px|alt=Ter sites in E. coli|Sequences of the ten ''Ter'' sites in ''E. coli'' with core sequences shaded in gray and the strictly conserved G-C(6) highlighted in yellow. Modified from <ref name="Duggin 2009" />.]]

===''Ter'' sites===

There are ten functional ''Ter'' sites (''TerA-J'') in ''E. coli'', each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all ''Ter'' sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of ''Ter'' is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved <ref name="Duggin 2009" /><ref name="Muclair 2006"> Mulcair, M.D., et al., A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell, 2006. 125(7): p. 1309-1319</ref>. These consensus sequences are important in Tus-''Ter'' complex formation.

<Structure load='1ECR' size='400' frame='true' align='right' caption='The Tus-Ter complex' scene='Insert optional scene name here' />

===Crystal structure of Tus when bound to ''Ter''===

The crystal structure of the Tus-''Ter'' complex was first unraveled by Kamada and co-workers <ref name="Kamada 1996" />. Tus is a 36 kDa protein that binds ''Ter'' as a monomer. It consists of two asymmetrical domains: a larger N-terminal and a smaller C-terminal domain. Each domain is made up of α-helices and β-sheets, linked by 4 loops (L1, L2, L3 and L4) that separate these secondary structures. ''Ter'' binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of ''Ter'' and intercalates tightly into the major groove of DNA. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated by [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bonds] (H-bonds) or [http://en.wikipedia.org/wiki/Van_der_Waals_forces van der Waal] interactions, as well as base contacts via H-bonds responsible for ''Ter'' recognition. Whilst the α-helical regions of both N- and C-terminal domains clamps the DNA, Tus is stabilized by high affinity binding of the interdomain.

===Mechanism of polar fork arrest===

Two models were proposed for halting replication fork movement at the non-permissive or blockage end <ref name="Kamada 1996" />:
#The “interaction” model
#The “clamp” model

====The "Interaction" model====
This model proposes the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-''Ter'' complex as it unwinds DNA in the 5’-3’ direction on the lagging strand during , protein-protein interactions between DnaB and Tus may be involved in replication fork arrest <ref name="Muclair 2006" />. In fact, based on site-directed mutagenesis studies demonstrates this special contact occurs at the L1 loop of Tus <ref name="Mulugu 2001"> Mulugu, S., et al., Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(17): p. 9569-9574</ref>. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end <ref name="Kaplan 2009" />.

====The "Clamp" model====
The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround ''Ter'' at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression <ref name="Kamada 1996" />. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.

Furthermore based on base substitution experiments of ''Ter'' sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of ''Ter'' - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket of Tus, forming a locked complex <ref name="Muclair 2006" /> which has a even higher binding affinity for ''Ter'' than normal double-stranded ''Ter'' binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Ter arrest mechanism fails <ref name="Kaplan 2009" />.

==In ''B. subtilis'': RTP-''Ter'' complex==

===''Ter'' sites===

9 Ter sites (I-IX)are arranged into two groups: 4 to arrest the clockwise replication fork and 5 for the anti-clockwise fork <ref name="vivian 2007">Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. Journal of Molecular Biology 370, 481-491, doi:10.1016/j.jmb.2007.02.067 (2007)</ref>.The Ter sites are 30 bp sequences comprising two imperfect inverted 16 bp repeats called the A and B sites <ref name="vivian 2007" />. The two sites overlap at a trinucleotide sequence that is highly conserved across all the Ter sites.

[[Image:B.Subtilis consensus Termination sequence.pngframe|alt=Ter site|B.Subtilis consensus Termination sequence showing A and B sites]]

Two homodimers of RTP bind to A and B sites cooperatively but the binding to the two sites are not equivalent. B site has a higher binding affinity to RTP dimer and has to be filled before A site <ref name="vivian 2007" />. The different hydrogen bonding sites and nonbonding contacts between the two sites and RTP were suggested to underlie the difference in binding affinity <ref name="wilce 2001">Wilce, J. A. et al. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Mol Biol 8, 206-210 (2001)</ref>. When both sites are filled, the affinity of RTP is higher due to cooperative binding interactions. A replication fork is only arrested when it is approaching from the non-permissive direction (proximal to B site).

===Crystal structure of RTP===

The first crystal structure presented by Bussiere et al suggested RTP binds to DNA as a symmetric homodimer of 29 kDa <ref name="Eli 1995">Eli Bussiere, D., Bastia, D. & Stephen White, W. Crystal structure of the replication terminator protein from ''B. subtiiis'' at 2.6 Å. Cell 80, 651-660, doi:Doi: 10.1016/0092-8674(95)90519-7 (1995)</ref>. This was further confirmed by structural study using RTP.C110S mutants in which the lone Cysteine residue is replaced by Serine and the adapted symmetric TerI B site (sRB) DNA that differ to native B site (nRB) by three bases <ref name="wilce 2001" />.However, a more recent study carried out by Vivian et al showed that crystal structure of the complex formed between RTP.C110S and the native nRB is asymmetric, consistent with an asymmetric binding mode in solution <ref name="vivian 2007" />.
RTP belongs to the α+β protein folding class and each monomer contains four α-helices, three β-sheets and an unstructured N-terminal <ref name="Eli 1995" />. It has the classic winged-helix motif where “wings” project up or down from the loop between β2-β3 strands <ref name="vivian 2007" /> and displays a unique mode of binding to DNA through interactions with both major and minor grooves <ref name="wilce 2001" />.The N-terminal halves of recognition (α3) helices of the dimer bind specifically to successive major grooves of DNA while the Theβ2-loop-β3 motif interacts with adjacent minor groove phosphate backbone <ref name="wilce 2001" />. The long C-terminal α helices (α4) of two monomers associate to form an antiparallel coiled-coil structure which defines the dimerisation domain <ref name="Eli 1995" />.

===Mechanism of polar fork arrest===

====RTP-DNA interaction====
As RTP was thought to bind to Ter site in a symmetrical conformation, the polarity of replication fork arrest was proposed to arise from the differential binding affinity and cooperative binding to A and B sites ('''Differential affinity model''')<ref name="vivian 2007" />. The binding of RTP to B site is strong enough to block replisome progression when A site is also filled whereas the weakly bound A site dimer will be displaced by advancing helicase <ref name="Duggin 2005" />. This displacement disrupts the cooperative binding between the two sites and hence the weakened RTP-B site complex will allow passage of the replication fork. Another model suggests that the binding of RTP to Ter site will result in significant conformational change, rendering the RTP-Ter complex structure asymmetric ('''conformational change model''')<ref name="vivian 2007" />. The two mechanisms are not mutually exclusive and may both account for the polarity of fork arrest. A recent crystal structure revealed that RTP is asymmetric in complex with DNA and the binding of RTP will result in distortion (bending) of upstream DNA <ref name="vivian 2007" />. This will facilitate further studies to provide insights into the basis of the polar action.

====RTP-replisome interaction====
In the past decade, a number of studies have demonstrated that the contacts between RTP and DNA is not sufficient to block replication fork progression, suggesting that asymmetric interactions between RTP and helicase is necessary for polar fork arrest <ref name="Kaplan 2009" />.Gautam et al showed that a very instable RTP-Ter complex at check point Ter site was able to arrest replication fork in vivo <ref name="Gautam 2001">Gautam, A. & Bastia, D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. Journal of Biological Chemistry 276, 8771-8777 (2001)</ref>. Duggin et al demonstrated that the ability of fork arrest does not strictly correlate to RTP-DNA binding affinity, suggesting a strong DNA-protein interaction is not obligatory for fork arrest <ref name="Duggin 2005 complex">Duggin, I. G., Matthews, J. M., Dixon, N. E., Wake, R. G. & Mackay, J. P. A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis. J. Biol. Chem. 280, 13105-13113, doi:10.1074/jbc.M414187200 (2005)</ref>. Another study revealed that fusion of peptides to the putative helicase interacting domain results in significantly reduced fork arresting efficiency without affecting DNA binding affinity <ref name="Duggin 2006">G. Duggin, I. DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding. Journal of Molecular Biology 361, 1-6, doi:DOI: 10.1016/j.jmb.2006.06.013 (2006)</ref>. It was also found that a mutant form of RTP with amino acid substitution at Tyr33 within the helix contact domain is not able to arrest replication fork, indicating the RTP-helicase interaction plays essential roles in mediating fork arrest <ref name="Gautam 2001 single domain">Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A Single Domain of the Replication Termination Protein of Bacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase. J. Biol. Chem. 276, 23471-23479, doi:10.1074/jbc.M009537200 (2001)</ref>.It is believed that interaction beween RTP and replisome is important for RTP function. This is achieved either the by '''highly specific surface residue interactions''' or '''secondary/tertiary structural complementarities''' <ref name="Gautam 2001">.

==Summary==
In summary, the termination sequences and replication terminator protein structures are dissimilar in ''E.coli'' and ''B.subtilis'', indicating independent origin of evolution. In spite of this, they both employ a replication fork trap mechanism in which high affinity binding of terminator protein to DNA through positively charged basic residues and terminator protein-replisome contact are required to achieve replication termination at defined terminus region in a polar manner.

==References==
<references />

File:Helicase approaching from B site.png

2011-05-21T06:51:38Z

Meng Han Liu: uploaded a new version of "Image:Helicase approaching from B site.png"

Graphic illustration of replication fork approching from B site of the Termination site (The non-permissive direction)