Proteopedia - User contributions [en]

User:Alexei Ilinykh/Sandbox 1

2011-05-23T03:11:31Z

Alexei Ilinykh: /* Evolution of Tus and RTP systems */

= Comparison of DNA Replication Termination in ''Bacillus subtilis'' and ''Escherichia coli'' =

== Termination sites in ''B.subtilis'' and ''E. coli'' ==
[[Image:EColi_chromosome_map.jpg|thumb|"border"|||300x300px|||Chromosome map of the 4.6 Mb ''E.Coli'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown.]]

Many bacteria that have a circular chromosome utilise a bidirectional mechanism of DNA replication <ref>PMID:12413665</ref>. Replication proceeds from the ''OriC'' site in two directions and terminate at some point ~180° away. DNA replication in Gram-negative ''Escherichia coli'' and Gram-positive ''Bacillus subtilis'' terminates at discrete Ter sites. Ter sites block the movement of the replication fork when the replication fork approaches from one direction (the non-permissive direction) but not the other (permissive direction). Ter sites stall replication forks only when bound by proteins Tus in ''E.Coli'' and Replication Termination Protein (RTP) in ''B.subtilis'' <ref>PMID:1444268</ref>. This molecular “trap” restricts the meeting of the replication forks to a small region of the chromosome and may be necessary for orderly segregation of the daughter strands <ref>PMID:19019156</ref>.

=== Ter sites in E.Coli ===

There are 10 Ter sites present in ''E.Coli'' <ref>PMID:9642188</ref>. ''TerC'', ''TerB'', ''TerF'', ''TerG'' and ''TerJ'' are oriented to block the clockwise movement of replication forks whilst ''TerA'', ''TerD'', ''TerE'', ''TerI'' and ''TerH'' block the anticlockwise movement of replication forks. Most of the replication forks are trapped in the region between ''TerC'' and ''TerA'' <ref>PMID:15738384</ref>. If the clockwise replication fork is delayed during DNA replication, termination occurs at ''TerA'' site. If the anticlockwise replication fork is delayed during DNA replication, termination occurs at ''TerC'' site.

Ter sites in E.Coli are 23 bp in length and are asymmetric. A comparison of all Ter sites revealed a consensus sequence (shown below) consisting of a highly conserved 13-bp core region and a strictly conserved GC6 base pair <ref>PMID:19298368</ref>. In the orientation shown, replication forks are blocked when travelling from the left and are unimpeded from the right. The nucleotides that interact with the Tus protein are shown in bold <ref>PMID:16148308</ref>.

[[Image:Ter_consensus_sequence.jpg||||"middle"|500x75px|||]]

=== Ter sites in ''B.subtilis'' ===
[[Image:Bsubtilis_chromosome_map.jpg|thumb|"border"|||500x350px|||Chromosome map of the 4.2 Mb ''B. Subtilis'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown]]

There are nine Ter sites present in ''B.subtilis'' <ref>PMID:9334221</ref>. ''TerIX'', ''TerV'', ''TerIII'' and ''TerI'' are oriented to block the clockwise movement of replication forks whilst ''TerII'', ''TerVIII'', TerIV, ''TerVII'' and ''TerVI'' block the anticlockwise movement of replication forks. In ''B.subtilis'', replication termination occurs mostly at ''TerI'' site because the anticlockwise replication fork takes longer to reach the termination region.

The ''TerI'' site is ~30 bp in length and consists of two overlapping half-sites: the core and auxiliary sequences <ref>PMID:2115089</ref>. The core sequence binds to RTP and this sequence-specific protein-DNA interaction allows the binding of a second RTP monomer to the auxiliary sequence <ref>PMID:1960722</ref>. The auxiliary sequence cannot bind to RTP in the absence of the core site. The ''TerI'' site is shown below with nucleotides involved in protein interactions highlighted in bold. In the orientation shown, replication forks are blocked when travelling from the right and are unimpeded from the left.

[[Image:Ter1_site.jpg||||"middle"|500x300px|||]]

=== Comparison of Ter sites in ''E.coli'' and ''B.subtilis'' ===

Both ''E.coli'' and ''B.subtilis'' have a similar number of Ter sites. However, the Ter sites do not show any sequence similarity <ref>PMID:1444268</ref>. The Ter sites in ''E.coli'' cover a large proportion of the genome with some sites closer to the origin of replication than the site of strand segregation. In contrast, the Ter sites in "B.subtilis" are clustered together in a small region of the chromosome close to the site of strand segregation.

== Structures of the Replication Termination Proteins ==

=== Tus ===

The Tus protein consists of 308 amino acids with a mass of 35.7 kDa and pI of 7.5 <ref>PMID:16148308</ref>. Tus forms a unique structural motif and has three distinct structural regions: two α-helical regions and a central β-sheet structure which forms the central cleft <ref>PMID:8857533</ref>. The protein is composed of 35% α-helix and 28% β-sheet. The '''secondary''' structure of the RTP molecule and its functions are listed in a table below:

[[Image:Tus_secondary_structure.jpg||||"middle"|500x2000px|||]]

Tus exists as a monomer in solution as shown by gel filtration experiments <ref>PMID:9334221</ref>.The tertiary structure of the protein can be seen in complex with DNA below (see section “Mechanisms of polar arrest”). A crystal structure on its own has not been isolated as yet. A number of important DNA-protein and protein-protein interactions are listed in a table below <ref>PMID:8857533</ref>:

[[Image:Tus_interactions.jpg||||"middle"|500x2000px|||]]

=== RTP ===
<Structure load='1BM9' size='350' frame='true' align='right' caption='Structure of RTP protein homodimer in solution' scene='Insert optional scene name here' />
RTP is a homodimer of subunit molecular weight of 14.5 kDa. The protein belongs to the “winged helix” family of proteins. Each monomer consists of 122 amino acid residues making up four α-helices, three β-strands, an extended loop and an unstructured N-terminal arm <ref>PMID:7867072</ref>. The protein is made of approximately 56% α-helix and 12% β-sheet. The '''secondary''' structure of the RTP molecule and its functions are listed in a table below:

[[Image:RTP_secondary_structure.jpg||||"middle"|500x2500px|||]]

The '''tertiary''' structure of RTP protein in solution is shown to the right <ref>PMID:7867072</ref>. A number of important DNA-protein <ref>PMID:17521668</ref> and protein-protein interactions are listed in a table below:

[[Image:RTP_inetractions.jpg||||"middle"|500x2500px|||]]

=== Comparison of Tus and RTP Protein Structures ===

Both Tus and RTP bind to their respective Ter sites on the bacterial chromosome and induce a direction-dependent replication fork arrest. However, rather surprisingly, the proteins do not have a common structural domain <ref>PMID:9016713</ref>. The Tus protein is a large asymmetric monomer that utilises mainly the β-sheet conformation to make specific interactions with DNA. In contrast, RTP binds to DNA as a symmetric homodimer and utilises mainly the α-helix conformation to make specific interactions with DNA.

In summary, whilst the two replication termination systems appear to share a common biological function, they do not share any DNA-binding sequence or structural homology. This strongly suggests an independent evolution of these systems.

== Mechanisms of Polar Arrest ==

=== RTP ===
<Structure load='2EFW' size='350' frame='true' align='right' caption='Structure of RTP complexed with a native RTP B site' scene='Insert optional scene name here' />

The ability of RTP to induce polar fork arrest in ''B. subtilis'' poses a problem, namely, how does a symmetrical dimer block the replication fork? Early analysis of the ''B. subtilis'' ter sites revealed two RTP binding sites; a high affinity core site (B site) and a low affinity auxiliary site (A site)<ref name = "terofDNArepchroandplas">PMID: 10209736</ref> . Mutagenic studies noted that generally contrahelicase activity was inversely correlated with binding affinity. However, some mutant forms were also found that showed no loss in DNA binding affinity despite a partial or complete loss in contrahelicase activity <ref name = "terofDNArepchroandplas" /> <ref>PMID: 8670817</ref>.

In contrast, initial studies of RTP suggested that polar fork arrest was more complex than just DNA binding affinity. Separate mutagenesis studies showed that contrahelicase activity was dependent on dimer-dimer interactions between RTP molecules bound to the ter site <ref>PMID: 8622923</ref>. Early studies of the protein structure predicted an asymmetrical complex would aid in the contrahelicase activity of RTP <ref>PMID: 7867072</ref>.

The issue of RTP and polar fork arrest was further complicated by Duggin ''et al.'' (2001) <ref>PMID: 11224562</ref>. Using X-ray crystallography and NMR spectroscopy, the group found RTP formed a symmetric complex with a synthetic symmetric RTP B site (sRB). It was found that in the symmetrical form, extensive dimer interactions caused an increase in DNA binding affinity. This led to the Differential Binding Affinity (DBA) model. In this model, polar fork arrest is generated from the different affinities of the B site and A sites within the ter sequence, caused by altered contact bases within the two sites.

However, a series of experiments displayed the DBA model to be inadequate in explaining polar fork arrest. Dixon ''et al''. (2005) showed sRB to be a poor terminator sequence ''in vitro'', and found no correlation between fork arrest activity and the affinity of the proximal binding site <ref>PMID: 15657033</ref>. Furthermore, Duggin (2006) showed that fusing peptides to the C-terminus of RTP could eliminate fork arrest activity, independent of size of amino acid sequence. The fusion of the peptide did not affect any DNA binding sites, though modelling suggests it would be able to contact the oncoming helicase <ref>PMID: 16822523</ref>.

Such lines of evidence led to a revaluation of the RTP-ter structure. Porter ''et al''. (2007) determined the crystal structure of RTP with a native RTP B binding site (nRB) <ref>PMID: 17521668</ref>. The paper found that the RTP dimer formed an asymmetric complex with nRB due to differences in downstream base contacts with the α3 recognition helix of RTP, creating a “wing-up”/”wing-down” dimer. Modelling of the structure showed the asymmetric complex would allow for extensive protein interactions between the RTP dimers. This underlies the cooperative binding that is crucial for fork arrest.

Taken together, these lines of evidence suggest that the polar fork arrest activity of RTP is dependent upon asymmetric contacts between RTP and the oncoming helicase, as mediated by the C-terminus <ref name = "mechofpolforkarr">PMID: 19298368</ref>.

===Tus===
<Structure load='2EWJ' size='350' frame='true' align='right' caption='Structure of the Tus-Ter B complex' scene='Insert optional scene name here' />

Despite the polar fork arrest activity of the Tus-ter complex in E. coli, fundamental differences exist between it and the RTP-ter system found in ''B. subtilis''. A basic difference is that Tus binds to ter as a monomer, rather than a dimer as is the case with RTP. This intrinsic asymmetry was predicted to be the basis of Tus’ polar fork arrest activity <ref name = "mechofpolforkarr" />. Furthermore, the crystal structures of RTP and Tus show vastly different tertiary protein folds and DNA binding motifs <ref name = "strofrepterprowithdna">PMID: 8857533</ref>.

Early studies of the Tus fork arrest mechanism led to the formation of two separate models; the “clamp model” and the “interactions model” <ref name = "repterinecoli">PMID: 16148308</ref>. Structural analysis appeared to support the “clamp model”, showing Tus would bind tightly to DNA and sterically impede the replication fork with the αIV and αV helices <ref name = "strofrepterprowithdna" />. However, some biochemical data seemed to indicate that a strong interaction occurred between Tus and the helicase. For instance, Bastia et al. (2001) showed that protein-protein interactions existed between Tus and DnaB using GST affinity chromatography and the yeast two-hybrid system. Furthermore, a series of mutants were identified with reduced Tus-DnaB interactions. These mutants were found to have substitutions within the L1 loop, and were shown to be deficient in fork arrest activity despite having higher DNA binding affinities <ref>PMID: 11493686</ref>. However, much of the biochemical data is inconsistent, and Tus has been known to block fork arrest of unrelated helicases in a polar manner, both supporting the clamp model <ref name = "repterinecoli" />.

Cross ''et al''. (2006) presented evidence for a modified clamp model <ref>PMID: 16814717</ref>. Using a series of forked Tus-ter complexes, the group was able to show that fork progression from the non-permissive face leads to an increase in the stability of the complex. Further analysis of the forked complexes identified the C (6) residue in ter to be responsible for the locking via a base flipping mechanism. X-ray crystallography found the cytosine residue flipped into a binding pocket where it could hydrogen bond to Gly 149, His 144 and Leu 150 as well as forming hydrophobic interactions with Ile 79 and Phe 140. This “molecular mousetrap” was proposed to be the basis of fork arrest on the non-permissive face of Tus. Doubly forked complexes showed Tus was removed from the complex as DNA contacts were removed as the fork progressed on the permissive side, thus accounting for polarity.

However, recent evidence suggests specific interactions between Tus and the oncoming helicase may be the basis of polar fork arrest. Using a sliding helicase lacking strand separation activity, Bastia ''et al''. (2008) showed Tus was able to block the sliding helicase on the non-permissive side even when the bases preceding C(6) were cross linked, and when the cytosine had been substituted. Hence, Tus was able to induce fork arrest without the base flipping mechanism. Furthermore, the group showed mutations in the L1 loop of Tus diminished its ability to arrest the sliding helicase <ref>PMID: 18708526</ref>.

Such evidence led to the establishment of a polar fork arrest model as seen in Bastia and Kaplan (2009) <ref name = "mechofpolforkarr" />. In this mechanism, a helicase approaching from the non-permissive face of the Tus-ter complex specifically interacts with Tus, whilst also being sterically impeded. In this mechanism, the base-flipping lock is used a fail-safe to ensure fork arrest. At the same time, a helicase approaching from the permissive face would be able to dislodge Tus due to the lack of protein-protein interactions.

=== Comparison between the mechanism of RTP and Tus ===
It is important to note that there is a degree of similarity between the polar arrest mechanisms of RTP and Tus. Both RTP and Tus require specific interactions with an oncoming helicase to induce fork arrest. In both cases, the polarity is established by a structural asymmetry. The idea that the two proteins have some basic mechanistic similarity is supported by the fact that RTP can arrest fork progression in ''E. coli'', as can Tus in ''B. subtilis'', albeit with far less efficiency <ref name = "mechofpolforkarr" />.

However, this asymmetry is accomplished in distinct ways; Tus is an inherently asymmetric monomer, whereas RTP takes on an asymmetric conformation once bound to DNA. RTP also lacks any specific base locking mechanism, as seen by the flipping of C (6) in the Tus-ter complex. The fact that such different proteins can have similar functions whilst sharing some basic similarities in mechanism supports the idea of independent, convergent evolution of the systems in ''E. coli'' and ''B. subtilis'' <ref name = "strofrepterprowithdna" />.

== Evolution of Tus and RTP systems ==

[[Image:phylogenetic.jpg|thumb|"border"|||300x300px||| Basic representations of the phylogenetic trees of E. coli and B. subtilis.]]

The study of Tus and the RTP systems reveal two systems which are key in the termination of replication in their hosts. However, while both systems induce termination, considerable differences in their structure and mechanisms of action strongly indicate that the systems evolved independently <ref>PMID:8857533</ref>.

Studies of the Tus-ter system have revealed the evolutionary development of the Tus protein in ''E. Coli'' <ref>PMID: 11523786</ref>. Although it has not been conserved identically in other species, there are organisms in which Tus-like proteins with high sequence homology have been identified <ref>PMID: 16148308</ref>. A study by Rocklein and Kuempel (1992) specifically identified ''S. enterica'' serovar Typhimurium as carrying a termination protein very similar to Tus with 78% sequence homology <ref>PMID: 1495392</ref>. More recent work has identified Tus-ter systems in ''Y. pestis'' and ''K. pneumonia'' both with sequence homologies over 50% <ref>PMID: 11523786</ref>. Furthermore, there have been studies showing Tus-like genes which have been found in plasmids. Specifically two proteins R394 and rts-1 have been found by Koch in two IncT plasmids <ref>PMID: 11106794</ref>.

Likewise, the RTP system of ''B. subtilius'' has many homologues in other species. Griffiths ''et al.'', (1998) found RTP homologs in ''B.atrophaeus'', ''b. amyloliquefaciens'', ''B mojavensis'' and ''B. vallismortis''. The sequences of RTP across the species were similar, but had up to 17.2% (''B. atrophaeus'') different bases. Yet despite this, only five amino acids were changed <ref>PMID: 9642188</ref>. This contrasts with Tus evolution, which has shown much greater sequence differences.

The research on the development of RTP in Bacillus utilized phylogenetic trees mapping the different species <ref>PMID: 11058132</ref>. This allowed the study to conclude that the RTP gene arose only after they had diverged from a common ancestor known as ''B. licheniformis'' <ref>PMID: 9642188</ref>. A more recent study, mapped the entire genome of ''B. licheniformis'' confirming that no RTP like gene existed. Furthermore, no RTP gene has been found on ''B. Halodurans'', narrowing the window in which RTP likely arose <ref>PMID: 15461803</ref>.

== The Dif site: The real termination point? ==
[[Image:Difsite.jpg|thumb|"border"|||500x350px|||Chromosome map of the 4.2 Mb ''B. Subtilis'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown along with the dif site]]

In bacteria, replication of DNA occurs by the way of two replication forks diverging away from the origin of replication, ''oriC'', before fusing and terminating at the terminus region. This system of replication is utilized in both ''E. Coli'' and ''B. Subtilis'' <ref>PMID: 10931283</ref>. These prokaryotes utilize two distinct termination systems, Tus and RTP respectively, to end replication. One interesting feature of replication in organisms with circular chromosomes, is the formation of chromosomal multimers. These multimers must be resolved through the use of Xer recombinases at a site called Dif. Without this, daughter cells cannot be segregated<ref>PMID: 21223577</ref>.

Discussion on the importance of the Dif site intensified after a study by Hendrickson and Lawrence (2007) suggested that it was the main site of replication termination <ref>PMID: 17376071</ref>. Prior to this, work suggested that numerous sites called Ter sites were the key to termination. These sites were arranged in a fashion such that they would “trap” and “arrest” termination in the terminus region <ref>PMID:10209736</ref>. However, Hendrickson and Lawrences' (2007) study utilized bioinformatics to suggest an alternative site of termination – the dif site. This inference was made by analysing G-C skew along the replication terminus. GC skew occurs because the lagging strand is more prone to mutation. Their cumulative plot of GC skewing demonstrated a peak where fork arrest occurred. The work by Hendrickson and Lawrence discovered such a peak very close to the dif site but not near any Ter site <ref>PMID: 17376071</ref>. Indeed, this finding held true in 8 different bacteria studied. While they did not directly suggest that the dif site was where termination occurred, they proposed a secondary mechanism whereby XerC/D recombinases binding to the dif site blocked the replication fork <ref>PMID: 17376071</ref>.

Duggin and Bell (2009) challenged the earlier findings and addressed these by utilizing 2D agarose gels to examine cells undergoing different stages of replication. Their findings showed clearly that there was fork arrest at ter sites and that these occurred at a reliable frequency that depended on their natural efficiency to induce fork arrest <ref>PMID: 19233209</ref>. Furthermore when they studied dif sites using a set of fragments of the dif region they found that there was no significant fork arrest anywhere near the dif region. Using these results they conclusively showed that the fork trap model with Ter sites was the most likely model of replication termination.

While the work performed in previous studies had studied bacteria, Duggin and Bell (2011) most recently performed a study on the Archaeon ''Sulfolobus solfataricus''. This Archea shares a degree of similiarity with the previously studied bacteria in possessing a circular chromosome with replication origins and also possessing dif sites. Importantly however, the paper showed that the dif site is not implicated in termination and furthermore exists outside fork fusion zones <ref>PMID: 21113132</ref>.

== References ==
<references/>

User:Alexei Ilinykh/Sandbox 1

2011-05-23T03:11:06Z

Alexei Ilinykh: /* Evolution of Tus and RTP systems */

= Comparison of DNA Replication Termination in ''Bacillus subtilis'' and ''Escherichia coli'' =

== Termination sites in ''B.subtilis'' and ''E. coli'' ==
[[Image:EColi_chromosome_map.jpg|thumb|"border"|||300x300px|||Chromosome map of the 4.6 Mb ''E.Coli'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown.]]

Many bacteria that have a circular chromosome utilise a bidirectional mechanism of DNA replication <ref>PMID:12413665</ref>. Replication proceeds from the ''OriC'' site in two directions and terminate at some point ~180° away. DNA replication in Gram-negative ''Escherichia coli'' and Gram-positive ''Bacillus subtilis'' terminates at discrete Ter sites. Ter sites block the movement of the replication fork when the replication fork approaches from one direction (the non-permissive direction) but not the other (permissive direction). Ter sites stall replication forks only when bound by proteins Tus in ''E.Coli'' and Replication Termination Protein (RTP) in ''B.subtilis'' <ref>PMID:1444268</ref>. This molecular “trap” restricts the meeting of the replication forks to a small region of the chromosome and may be necessary for orderly segregation of the daughter strands <ref>PMID:19019156</ref>.

=== Ter sites in E.Coli ===

There are 10 Ter sites present in ''E.Coli'' <ref>PMID:9642188</ref>. ''TerC'', ''TerB'', ''TerF'', ''TerG'' and ''TerJ'' are oriented to block the clockwise movement of replication forks whilst ''TerA'', ''TerD'', ''TerE'', ''TerI'' and ''TerH'' block the anticlockwise movement of replication forks. Most of the replication forks are trapped in the region between ''TerC'' and ''TerA'' <ref>PMID:15738384</ref>. If the clockwise replication fork is delayed during DNA replication, termination occurs at ''TerA'' site. If the anticlockwise replication fork is delayed during DNA replication, termination occurs at ''TerC'' site.

Ter sites in E.Coli are 23 bp in length and are asymmetric. A comparison of all Ter sites revealed a consensus sequence (shown below) consisting of a highly conserved 13-bp core region and a strictly conserved GC6 base pair <ref>PMID:19298368</ref>. In the orientation shown, replication forks are blocked when travelling from the left and are unimpeded from the right. The nucleotides that interact with the Tus protein are shown in bold <ref>PMID:16148308</ref>.

[[Image:Ter_consensus_sequence.jpg||||"middle"|500x75px|||]]

=== Ter sites in ''B.subtilis'' ===
[[Image:Bsubtilis_chromosome_map.jpg|thumb|"border"|||500x350px|||Chromosome map of the 4.2 Mb ''B. Subtilis'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown]]

There are nine Ter sites present in ''B.subtilis'' <ref>PMID:9334221</ref>. ''TerIX'', ''TerV'', ''TerIII'' and ''TerI'' are oriented to block the clockwise movement of replication forks whilst ''TerII'', ''TerVIII'', TerIV, ''TerVII'' and ''TerVI'' block the anticlockwise movement of replication forks. In ''B.subtilis'', replication termination occurs mostly at ''TerI'' site because the anticlockwise replication fork takes longer to reach the termination region.

The ''TerI'' site is ~30 bp in length and consists of two overlapping half-sites: the core and auxiliary sequences <ref>PMID:2115089</ref>. The core sequence binds to RTP and this sequence-specific protein-DNA interaction allows the binding of a second RTP monomer to the auxiliary sequence <ref>PMID:1960722</ref>. The auxiliary sequence cannot bind to RTP in the absence of the core site. The ''TerI'' site is shown below with nucleotides involved in protein interactions highlighted in bold. In the orientation shown, replication forks are blocked when travelling from the right and are unimpeded from the left.

[[Image:Ter1_site.jpg||||"middle"|500x300px|||]]

=== Comparison of Ter sites in ''E.coli'' and ''B.subtilis'' ===

Both ''E.coli'' and ''B.subtilis'' have a similar number of Ter sites. However, the Ter sites do not show any sequence similarity <ref>PMID:1444268</ref>. The Ter sites in ''E.coli'' cover a large proportion of the genome with some sites closer to the origin of replication than the site of strand segregation. In contrast, the Ter sites in "B.subtilis" are clustered together in a small region of the chromosome close to the site of strand segregation.

== Structures of the Replication Termination Proteins ==

=== Tus ===

The Tus protein consists of 308 amino acids with a mass of 35.7 kDa and pI of 7.5 <ref>PMID:16148308</ref>. Tus forms a unique structural motif and has three distinct structural regions: two α-helical regions and a central β-sheet structure which forms the central cleft <ref>PMID:8857533</ref>. The protein is composed of 35% α-helix and 28% β-sheet. The '''secondary''' structure of the RTP molecule and its functions are listed in a table below:

[[Image:Tus_secondary_structure.jpg||||"middle"|500x2000px|||]]

Tus exists as a monomer in solution as shown by gel filtration experiments <ref>PMID:9334221</ref>.The tertiary structure of the protein can be seen in complex with DNA below (see section “Mechanisms of polar arrest”). A crystal structure on its own has not been isolated as yet. A number of important DNA-protein and protein-protein interactions are listed in a table below <ref>PMID:8857533</ref>:

[[Image:Tus_interactions.jpg||||"middle"|500x2000px|||]]

=== RTP ===
<Structure load='1BM9' size='350' frame='true' align='right' caption='Structure of RTP protein homodimer in solution' scene='Insert optional scene name here' />
RTP is a homodimer of subunit molecular weight of 14.5 kDa. The protein belongs to the “winged helix” family of proteins. Each monomer consists of 122 amino acid residues making up four α-helices, three β-strands, an extended loop and an unstructured N-terminal arm <ref>PMID:7867072</ref>. The protein is made of approximately 56% α-helix and 12% β-sheet. The '''secondary''' structure of the RTP molecule and its functions are listed in a table below:

[[Image:RTP_secondary_structure.jpg||||"middle"|500x2500px|||]]

The '''tertiary''' structure of RTP protein in solution is shown to the right <ref>PMID:7867072</ref>. A number of important DNA-protein <ref>PMID:17521668</ref> and protein-protein interactions are listed in a table below:

[[Image:RTP_inetractions.jpg||||"middle"|500x2500px|||]]

=== Comparison of Tus and RTP Protein Structures ===

Both Tus and RTP bind to their respective Ter sites on the bacterial chromosome and induce a direction-dependent replication fork arrest. However, rather surprisingly, the proteins do not have a common structural domain <ref>PMID:9016713</ref>. The Tus protein is a large asymmetric monomer that utilises mainly the β-sheet conformation to make specific interactions with DNA. In contrast, RTP binds to DNA as a symmetric homodimer and utilises mainly the α-helix conformation to make specific interactions with DNA.

In summary, whilst the two replication termination systems appear to share a common biological function, they do not share any DNA-binding sequence or structural homology. This strongly suggests an independent evolution of these systems.

== Mechanisms of Polar Arrest ==

=== RTP ===
<Structure load='2EFW' size='350' frame='true' align='right' caption='Structure of RTP complexed with a native RTP B site' scene='Insert optional scene name here' />

The ability of RTP to induce polar fork arrest in ''B. subtilis'' poses a problem, namely, how does a symmetrical dimer block the replication fork? Early analysis of the ''B. subtilis'' ter sites revealed two RTP binding sites; a high affinity core site (B site) and a low affinity auxiliary site (A site)<ref name = "terofDNArepchroandplas">PMID: 10209736</ref> . Mutagenic studies noted that generally contrahelicase activity was inversely correlated with binding affinity. However, some mutant forms were also found that showed no loss in DNA binding affinity despite a partial or complete loss in contrahelicase activity <ref name = "terofDNArepchroandplas" /> <ref>PMID: 8670817</ref>.

In contrast, initial studies of RTP suggested that polar fork arrest was more complex than just DNA binding affinity. Separate mutagenesis studies showed that contrahelicase activity was dependent on dimer-dimer interactions between RTP molecules bound to the ter site <ref>PMID: 8622923</ref>. Early studies of the protein structure predicted an asymmetrical complex would aid in the contrahelicase activity of RTP <ref>PMID: 7867072</ref>.

The issue of RTP and polar fork arrest was further complicated by Duggin ''et al.'' (2001) <ref>PMID: 11224562</ref>. Using X-ray crystallography and NMR spectroscopy, the group found RTP formed a symmetric complex with a synthetic symmetric RTP B site (sRB). It was found that in the symmetrical form, extensive dimer interactions caused an increase in DNA binding affinity. This led to the Differential Binding Affinity (DBA) model. In this model, polar fork arrest is generated from the different affinities of the B site and A sites within the ter sequence, caused by altered contact bases within the two sites.

However, a series of experiments displayed the DBA model to be inadequate in explaining polar fork arrest. Dixon ''et al''. (2005) showed sRB to be a poor terminator sequence ''in vitro'', and found no correlation between fork arrest activity and the affinity of the proximal binding site <ref>PMID: 15657033</ref>. Furthermore, Duggin (2006) showed that fusing peptides to the C-terminus of RTP could eliminate fork arrest activity, independent of size of amino acid sequence. The fusion of the peptide did not affect any DNA binding sites, though modelling suggests it would be able to contact the oncoming helicase <ref>PMID: 16822523</ref>.

Such lines of evidence led to a revaluation of the RTP-ter structure. Porter ''et al''. (2007) determined the crystal structure of RTP with a native RTP B binding site (nRB) <ref>PMID: 17521668</ref>. The paper found that the RTP dimer formed an asymmetric complex with nRB due to differences in downstream base contacts with the α3 recognition helix of RTP, creating a “wing-up”/”wing-down” dimer. Modelling of the structure showed the asymmetric complex would allow for extensive protein interactions between the RTP dimers. This underlies the cooperative binding that is crucial for fork arrest.

Taken together, these lines of evidence suggest that the polar fork arrest activity of RTP is dependent upon asymmetric contacts between RTP and the oncoming helicase, as mediated by the C-terminus <ref name = "mechofpolforkarr">PMID: 19298368</ref>.

===Tus===
<Structure load='2EWJ' size='350' frame='true' align='right' caption='Structure of the Tus-Ter B complex' scene='Insert optional scene name here' />

Despite the polar fork arrest activity of the Tus-ter complex in E. coli, fundamental differences exist between it and the RTP-ter system found in ''B. subtilis''. A basic difference is that Tus binds to ter as a monomer, rather than a dimer as is the case with RTP. This intrinsic asymmetry was predicted to be the basis of Tus’ polar fork arrest activity <ref name = "mechofpolforkarr" />. Furthermore, the crystal structures of RTP and Tus show vastly different tertiary protein folds and DNA binding motifs <ref name = "strofrepterprowithdna">PMID: 8857533</ref>.

Early studies of the Tus fork arrest mechanism led to the formation of two separate models; the “clamp model” and the “interactions model” <ref name = "repterinecoli">PMID: 16148308</ref>. Structural analysis appeared to support the “clamp model”, showing Tus would bind tightly to DNA and sterically impede the replication fork with the αIV and αV helices <ref name = "strofrepterprowithdna" />. However, some biochemical data seemed to indicate that a strong interaction occurred between Tus and the helicase. For instance, Bastia et al. (2001) showed that protein-protein interactions existed between Tus and DnaB using GST affinity chromatography and the yeast two-hybrid system. Furthermore, a series of mutants were identified with reduced Tus-DnaB interactions. These mutants were found to have substitutions within the L1 loop, and were shown to be deficient in fork arrest activity despite having higher DNA binding affinities <ref>PMID: 11493686</ref>. However, much of the biochemical data is inconsistent, and Tus has been known to block fork arrest of unrelated helicases in a polar manner, both supporting the clamp model <ref name = "repterinecoli" />.

Cross ''et al''. (2006) presented evidence for a modified clamp model <ref>PMID: 16814717</ref>. Using a series of forked Tus-ter complexes, the group was able to show that fork progression from the non-permissive face leads to an increase in the stability of the complex. Further analysis of the forked complexes identified the C (6) residue in ter to be responsible for the locking via a base flipping mechanism. X-ray crystallography found the cytosine residue flipped into a binding pocket where it could hydrogen bond to Gly 149, His 144 and Leu 150 as well as forming hydrophobic interactions with Ile 79 and Phe 140. This “molecular mousetrap” was proposed to be the basis of fork arrest on the non-permissive face of Tus. Doubly forked complexes showed Tus was removed from the complex as DNA contacts were removed as the fork progressed on the permissive side, thus accounting for polarity.

However, recent evidence suggests specific interactions between Tus and the oncoming helicase may be the basis of polar fork arrest. Using a sliding helicase lacking strand separation activity, Bastia ''et al''. (2008) showed Tus was able to block the sliding helicase on the non-permissive side even when the bases preceding C(6) were cross linked, and when the cytosine had been substituted. Hence, Tus was able to induce fork arrest without the base flipping mechanism. Furthermore, the group showed mutations in the L1 loop of Tus diminished its ability to arrest the sliding helicase <ref>PMID: 18708526</ref>.

Such evidence led to the establishment of a polar fork arrest model as seen in Bastia and Kaplan (2009) <ref name = "mechofpolforkarr" />. In this mechanism, a helicase approaching from the non-permissive face of the Tus-ter complex specifically interacts with Tus, whilst also being sterically impeded. In this mechanism, the base-flipping lock is used a fail-safe to ensure fork arrest. At the same time, a helicase approaching from the permissive face would be able to dislodge Tus due to the lack of protein-protein interactions.

=== Comparison between the mechanism of RTP and Tus ===
It is important to note that there is a degree of similarity between the polar arrest mechanisms of RTP and Tus. Both RTP and Tus require specific interactions with an oncoming helicase to induce fork arrest. In both cases, the polarity is established by a structural asymmetry. The idea that the two proteins have some basic mechanistic similarity is supported by the fact that RTP can arrest fork progression in ''E. coli'', as can Tus in ''B. subtilis'', albeit with far less efficiency <ref name = "mechofpolforkarr" />.

However, this asymmetry is accomplished in distinct ways; Tus is an inherently asymmetric monomer, whereas RTP takes on an asymmetric conformation once bound to DNA. RTP also lacks any specific base locking mechanism, as seen by the flipping of C (6) in the Tus-ter complex. The fact that such different proteins can have similar functions whilst sharing some basic similarities in mechanism supports the idea of independent, convergent evolution of the systems in ''E. coli'' and ''B. subtilis'' <ref name = "strofrepterprowithdna" />.

== Evolution of Tus and RTP systems ==

[[Image:EColi_chromosome_map.jpg|thumb|"border"|||300x300px||| Basic representations of the phylogenetic trees of E. coli and B. subtilis.]]

The study of Tus and the RTP systems reveal two systems which are key in the termination of replication in their hosts. However, while both systems induce termination, considerable differences in their structure and mechanisms of action strongly indicate that the systems evolved independently <ref>PMID:8857533</ref>.

Studies of the Tus-ter system have revealed the evolutionary development of the Tus protein in ''E. Coli'' <ref>PMID: 11523786</ref>. Although it has not been conserved identically in other species, there are organisms in which Tus-like proteins with high sequence homology have been identified <ref>PMID: 16148308</ref>. A study by Rocklein and Kuempel (1992) specifically identified ''S. enterica'' serovar Typhimurium as carrying a termination protein very similar to Tus with 78% sequence homology <ref>PMID: 1495392</ref>. More recent work has identified Tus-ter systems in ''Y. pestis'' and ''K. pneumonia'' both with sequence homologies over 50% <ref>PMID: 11523786</ref>. Furthermore, there have been studies showing Tus-like genes which have been found in plasmids. Specifically two proteins R394 and rts-1 have been found by Koch in two IncT plasmids <ref>PMID: 11106794</ref>.

Likewise, the RTP system of ''B. subtilius'' has many homologues in other species. Griffiths ''et al.'', (1998) found RTP homologs in ''B.atrophaeus'', ''b. amyloliquefaciens'', ''B mojavensis'' and ''B. vallismortis''. The sequences of RTP across the species were similar, but had up to 17.2% (''B. atrophaeus'') different bases. Yet despite this, only five amino acids were changed <ref>PMID: 9642188</ref>. This contrasts with Tus evolution, which has shown much greater sequence differences.

The research on the development of RTP in Bacillus utilized phylogenetic trees mapping the different species <ref>PMID: 11058132</ref>. This allowed the study to conclude that the RTP gene arose only after they had diverged from a common ancestor known as ''B. licheniformis'' <ref>PMID: 9642188</ref>. A more recent study, mapped the entire genome of ''B. licheniformis'' confirming that no RTP like gene existed. Furthermore, no RTP gene has been found on ''B. Halodurans'', narrowing the window in which RTP likely arose <ref>PMID: 15461803</ref>.

== The Dif site: The real termination point? ==
[[Image:Difsite.jpg|thumb|"border"|||500x350px|||Chromosome map of the 4.2 Mb ''B. Subtilis'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown along with the dif site]]

In bacteria, replication of DNA occurs by the way of two replication forks diverging away from the origin of replication, ''oriC'', before fusing and terminating at the terminus region. This system of replication is utilized in both ''E. Coli'' and ''B. Subtilis'' <ref>PMID: 10931283</ref>. These prokaryotes utilize two distinct termination systems, Tus and RTP respectively, to end replication. One interesting feature of replication in organisms with circular chromosomes, is the formation of chromosomal multimers. These multimers must be resolved through the use of Xer recombinases at a site called Dif. Without this, daughter cells cannot be segregated<ref>PMID: 21223577</ref>.

Discussion on the importance of the Dif site intensified after a study by Hendrickson and Lawrence (2007) suggested that it was the main site of replication termination <ref>PMID: 17376071</ref>. Prior to this, work suggested that numerous sites called Ter sites were the key to termination. These sites were arranged in a fashion such that they would “trap” and “arrest” termination in the terminus region <ref>PMID:10209736</ref>. However, Hendrickson and Lawrences' (2007) study utilized bioinformatics to suggest an alternative site of termination – the dif site. This inference was made by analysing G-C skew along the replication terminus. GC skew occurs because the lagging strand is more prone to mutation. Their cumulative plot of GC skewing demonstrated a peak where fork arrest occurred. The work by Hendrickson and Lawrence discovered such a peak very close to the dif site but not near any Ter site <ref>PMID: 17376071</ref>. Indeed, this finding held true in 8 different bacteria studied. While they did not directly suggest that the dif site was where termination occurred, they proposed a secondary mechanism whereby XerC/D recombinases binding to the dif site blocked the replication fork <ref>PMID: 17376071</ref>.

Duggin and Bell (2009) challenged the earlier findings and addressed these by utilizing 2D agarose gels to examine cells undergoing different stages of replication. Their findings showed clearly that there was fork arrest at ter sites and that these occurred at a reliable frequency that depended on their natural efficiency to induce fork arrest <ref>PMID: 19233209</ref>. Furthermore when they studied dif sites using a set of fragments of the dif region they found that there was no significant fork arrest anywhere near the dif region. Using these results they conclusively showed that the fork trap model with Ter sites was the most likely model of replication termination.

While the work performed in previous studies had studied bacteria, Duggin and Bell (2011) most recently performed a study on the Archaeon ''Sulfolobus solfataricus''. This Archea shares a degree of similiarity with the previously studied bacteria in possessing a circular chromosome with replication origins and also possessing dif sites. Importantly however, the paper showed that the dif site is not implicated in termination and furthermore exists outside fork fusion zones <ref>PMID: 21113132</ref>.

== References ==
<references/>

2011-05-22T08:36:47Z

Alexei Ilinykh:

User:Alexei Ilinykh/Sandbox 1

2011-05-22T08:33:35Z

Alexei Ilinykh:

== Termination sites in ''B.subtilis'' and ''E. coli'' ==
[[Image:EColi_chromosome_map.jpg|thumb|"border"|||300x300px|||Chromosome map of the 4.6 Mb ''E.Coli'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown.]]

Many bacteria that have a circular chromosome utilise a bidirectional mechanism of DNA replication (Messer, 2002). Replication proceeds from the ''OriC'' site in two directions and terminate at some point ~180° away. DNA replication in Gram-negative ''Escherichia coli'' and Gram-positive ''Bacillus subtilis'' terminates at discrete Ter sites. Ter sites block the movement of the replication fork when the replication fork approaches from one direction (the non-permissive direction) but not the other (permissive direction). Ter sites stall replication forks only when bound by proteins Tus in ''E.Coli'' and Replication Termination Protein (RTP) in ''B.subtilis'' (Hill, 1992). This molecular “trap” restricts the meeting of the replication forks to a small region of the chromosome and may be necessary for orderly segregation of the daughter strands (Duggin et al., 2008).

=== Ter sites in E.Coli ===

There are 10 Ter sites present in ''E.Coli'' (Griffiths et al., 1998). ''TerC'', ''TerB'', ''TerF'', ''TerG'' and ''TerJ'' are oriented to block the clockwise movement of replication forks whilst ''TerA'', ''TerD'', ''TerE'', ''TerI'' and ''TerH'' block the anticlockwise movement of replication forks. Most of the replication forks are trapped in the region between ''TerC'' and ''TerA'' (Breier et al., 2005). If the clockwise replication fork is delayed during DNA replication, termination occurs at ''TerA'' site. If the anticlockwise replication fork is delayed during DNA replication, termination occurs at ''TerC'' site.

Ter sites in E.Coli are 23 bp in length and are asymmetric. A comparison of all Ter sites revealed a consensus sequence (shown below) consisting of a highly conserved 13-bp core region and a strictly conserved GC6 base pair (Kaplan and Bastia, 2009). In the orientation shown, replication forks are blocked when travelling from the left and are unimpeded from the right. The nucleotides that interact with the Tus protein are shown in bold (Neylon et al., 2005)

[[Image:Ter_consensus_sequence.jpg||||"middle"|500x75px|||]]

=== Ter sites in ''B.subtilis'' ===
[[Image:Bsubtilis_chromosome_map.jpg|thumb|"border"|||500x350px|||Chromosome map of the 4.2 Mb ''B. Subtilis'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown]]

There are nine Ter sites present in ''B.subtilis'' (Coskun-Ari and Hill, 1997). ''TerIX'', ''TerV'', ''TerIII'' and ''TerI'' are oriented to block the clockwise movement of replication forks whilst ''TerII'', ''TerVIII'', TerIV, ''TerVII'' and ''TerVI'' block the anticlockwise movement of replication forks. In ''B.subtilis'', replication termination occurs mostly at ''TerI'' site because the anticlockwise replication fork takes longer to reach the termination region.

The ''TerI'' site is ~30 bp in length and consists of two overlapping half-sites: the core and auxiliary sequences (Lewis et al., 1990). The core sequence binds to RTP and this sequence-specific protein-DNA interaction allows the binding of a second RTP monomer to the auxiliary sequence (Carrigan et al., 1991). The auxiliary sequence cannot bind to RTP in the absence of the core site. The ''TerI'' site is shown below with nucleotides involved in protein interactions highlighted in bold. In the orientation shown, replication forks are blocked when travelling from the right and are unimpeded from the left.

[[Image:Ter1_site.jpg||||"middle"|500x300px|||]]

=== Comparison of Ter sites in ''E.coli'' and ''B.subtilis'' ===

Both ''E.coli'' and ''B.subtilis'' have a similar number of Ter sites. However, the Ter sites do not show any sequence similarity (Hill, 1992). The Ter sites in ''E.coli'' cover a large proportion of the genome with some sites closer to the origin of replication than the site of strand segregation. In contrast, the Ter sites in "B.subtilis" are clustered together in a small region of the chromosome close to the site of strand segregation.

== Structures of the Replication Termination Proteins ==

=== Tus ===

The Tus protein consists of 308 amino acids with a mass of 35.7 kDa and pI of 7.5 (Neylon et al., 2005). Tus forms a unique structural motif and has three distinct structural regions: two α-helical regions and a central β-sheet structure which forms the central cleft (Kamada et al., 1996). The protein is composed of 35% α-helix and 28% β-sheet. The '''secondary''' structure of the RTP molecule and its functions are listed in a table below:

[[Image:Tus_secondary_structure.jpg||||"middle"|500x2000px|||]]

Tus exists as a monomer in solution as shown by gel filtration experiments (Coskun-Ari et al., 1994).The tertiary structure of the protein can be seen in complex with DNA below (see section “Mechanisms of polar arrest”). A crystal structure on its own has not been isolated as yet. A number of important DNA-protein and protein-protein interactions are listed in a table below (Kamada et al., 1996):

[[Image:Tus_interactions.jpg||||"middle"|500x2000px|||]]

=== RTP ===
<Structure load='1BM9' size='350' frame='true' align='right' caption='Structure of RTP protein homodimer in solution' scene='Insert optional scene name here' />
RTP is a homodimer of subunit molecular weight of 14.5 kDa. The protein belongs to the “winged helix” family of proteins. Each monomer consists of 122 amino acid residues making up four α-helices, three β-strands, an extended loop and an unstructured N-terminal arm (Bussiere et al., 1995). The protein is made of approximately 56% α-helix and 12% β-sheet. The '''secondary''' structure of the RTP molecule and its functions are listed in a table below:

[[Image:RTP_secondary_structure.jpg||||"middle"|500x2500px|||]]

The '''tertiary''' structure of RTP protein in solution is shown to the right (Bussiere et al., 1995). A number of important DNA-protein (Vivian et al., 2007) and protein-protein interactions are listed in a table below:

[[Image:RTP_inetractions.jpg||||"middle"|500x2500px|||]]

== Mechanisms of Polar Arrest ==

=== RTP ===
<Structure load='2EFW' size='350' frame='true' align='right' caption='Structure of RTP complexed with a native RTP B site' scene='Insert optional scene name here' />

The ability of RTP to induce polar fork arrest in ''B. subtilis'' poses a problem, namely, how does a symmetrical dimer block the replication fork? Early analysis of the ''B. subtilis'' ter sites revealed two RTP binding sites; a high affinity core site (B site) and a low affinity auxiliary site (A site)<ref name = "terofDNArepchroandplas">PMID: 10209736</ref> . Mutagenic studies noted that generally contrahelicase activity was inversely correlated with binding affinity. However, some mutant forms were also found that showed no loss in DNA binding affinity despite a partial or complete loss in contrahelicase activity <ref name = "terofDNArepchroandplas" /> <ref>PMID: 8670817</ref>.

In contrast, initial studies of RTP suggested that polar fork arrest was more complex than just DNA binding affinity. Separate mutagenesis studies showed that contrahelicase activity was dependent on dimer-dimer interactions between RTP molecules bound to the ter site <ref>PMID: 8622923</ref>. Early studies of the protein structure predicted an asymmetrical complex would aid in the contrahelicase activity of RTP <ref>PMID: 7867072</ref>.

The issue of RTP and polar fork arrest was further complicated by Duggin ''et al.'' (2001) <ref>PMID: 11224562</ref>. Using X-ray crystallography and NMR spectroscopy, the group found RTP formed a symmetric complex with a synthetic symmetric RTP B site (sRB). It was found that in the symmetrical form, extensive dimer interactions caused an increase in DNA binding affinity. This led to the Differential Binding Affinity (DBA) model. In this model, polar fork arrest is generated from the different affinities of the B site and A sites within the ter sequence, caused by altered contact bases within the two sites.

However, a series of experiments displayed the DBA model to be inadequate in explaining polar fork arrest. Dixon ''et al''. (2005) showed sRB to be a poor terminator sequence ''in vitro'', and found no correlation between fork arrest activity and the affinity of the proximal binding site <ref>PMID: 15657033</ref>. Furthermore, Duggin (2006) showed that fusing peptides to the C-terminus of RTP could eliminate fork arrest activity, independent of size of amino acid sequence. The fusion of the peptide did not affect any DNA binding sites, but rather an area of RTP that was proposed to contact the oncoming helicase <ref>PMID: 16822523</ref>.

Such lines of evidence led to a revaluation of the RTP-ter structure. Porter ''et al''. (2007) determined the crystal structure of RTP with a native RTP B binding site (nRB) <ref>PMID: 17521668</ref>. The paper found that the RTP dimer formed an asymmetric complex with nRB due to differences in downstream base contacts with the α3 recognition helix of RTP, creating a “wing-up”/”wing-down” dimer. Modelling of the structure showed the asymmetric complex would allow for extensive protein interactions between the RTP dimers. This underlies the cooperative binding that is crucial for fork arrest.

Taken together, these lines of evidence suggest that the polar fork arrest activity of RTP is dependent upon asymmetric contacts between RTP and the oncoming helicase, as mediated by the C-terminus <ref name = "mechofpolforkarr">PMID: 19298368</ref>.

===Tus===
<Structure load='2EWJ' size='350' frame='true' align='right' caption='Structure of the Tus-''Ter''B complex' scene='Insert optional scene name here' />

Despite the polar fork arrest activity of the Tus-ter complex in E. coli, fundamental differences exist between it and the RTP-ter system found in ''B. subtilis''. A basic difference is that Tus binds to ter as a monomer, rather than a dimer as is the case with RTP. This intrinsic asymmetry was predicted to be the basis of Tus’ polar fork arrest activity <ref name = "mechofpolforkarr" />. Furthermore, the crystal structures of RTP and Tus show vastly different tertiary protein folds and DNA binding motifs <ref name = "strofrepterprowithdna">PMID: 8857533</ref>.

Early studies of the Tus fork arrest mechanism led to the formation of two separate models; the “clamp model” and the “interactions model” <ref name = "repterinecoli">PMID: 16148308</ref>. Structural analysis appeared to support the “clamp model”, showing Tus would bind tightly to DNA and sterically impede the replication fork with the αIV and αV helices <ref name = "strofrepterprowithdna" />. However, some biochemical data seemed to indicate that a strong interaction occurred between Tus and the helicase. For instance, Bastia et al. (2001) showed that protein-protein interactions existed between Tus and DnaB using GST affinity chromatography and the yeast two-hybrid system. Furthermore, a series of mutants were identified with reduced Tus-DnaB interactions. These mutants were found to have substitutions within the L1 loop, and were shown to be deficient in fork arrest activity despite having higher DNA binding affinities <ref>PMID: 11493686</ref>. However, much of the biochemical data is inconsistent, and Tus has been known to block fork arrest of unrelated helicases in a polar manner, both supporting the clamp model <ref name = "repterinecoli" />.

Cross ''et al''. (2006) presented evidence for a modified clamp model <ref>PMID: 16814717</ref>. Using a series of forked Tus-ter complexes, the group was able to show that fork progression from the non-permissive face leads to an increase in the stability of the complex. Further analysis of the forked complexes identified the C (6) residue in ter to be responsible for the locking via a base flipping mechanism. X-ray crystallography found the cytosine residue flipped into a binding pocket where it could hydrogen bond to Gly 149, His 144 and Leu 150 as well as forming hydrophobic interactions with Ile 79 and Phe 140. This “molecular mousetrap” was proposed to be the basis of fork arrest on the non-permissive face of Tus. Doubly forked complexes showed Tus was removed from the complex as DNA contacts were removed as the fork progressed on the permissive side, thus accounting for polarity.

However, recent evidence suggests specific interactions between Tus and the oncoming helicase may be the basis of polar fork arrest. Using a sliding helicase lacking strand separation activity, Bastia ''et al''. (2008) showed Tus was able to block the sliding helicase on the non-permissive side even when the bases preceding C(6) were cross linked, and when the cytosine had been substituted. Hence, Tus was able to induce fork arrest without the base flipping mechanism. Furthermore, the group showed mutations in the L1 loop of Tus diminished its ability to arrest the sliding helicase <ref>PMID: 18708526</ref>.

Such evidence led to the establishment of a polar fork arrest model as seen in Bastia and Kaplan (2009) <ref name = "mechofpolforkarr" />. In this mechanism, a helicase approaching from the non-permissive face of the Tus-ter complex specifically interacts with Tus, whilst also being sterically impeded. In this mechanism, the base-flipping lock is used a fail-safe to ensure fork arrest. At the same time, a helicase approaching from the permissive face would be able to dislodge Tus due to the lack of protein-protein interactions.

=== Comparison between the mechanism of RTP and Tus ===
It is important to note that there is a degree of similarity between the polar arrest mechanisms of RTP and Tus, in so far that specific interactions with an oncoming helicase is required for its impediment. In both cases, the polarity is established by a structural asymmetry. The idea that the two proteins have some basic mechanistic similarity is supported by the fact that RTP can arrest fork progression in ''E. coli'', as can Tus in ''B. subtilis'', albeit with far less efficiency <ref name = "mechofpolforkarr" />.

However, this asymmetry is accomplished in distinct ways; Tus is an inherently asymmetric monomer, whereas RTP takes on an asymmetric conformation once bound to DNA. RTP also lacks any specific base locking mechanism, as seen by the flipping of C (6) in the Tus-ter complex. The fact that such different proteins can have similar functions whilst sharing some basic similarities in mechanism supports the idea of independent, convergent evolution of the systems in ''E. coli'' and ''B. subtilis'' <ref name = "strofrepterprowithdna" />.

== The Dif site: The real termination point? ==

In bacteria, replication of DNA occurs by the way of two replication forks diverging away from the origin of replication, ''oriC'', before fusing and terminating at the terminus region. This system of replication is utilized in both ''E. Coli'' and ''B. Subtilis'' <ref>PMID: 10931283</ref>. These prokaryotes utilize two distinct termination systems, Tus and RTP respectively, to end replication. One interesting feature of replication in organisms with circular chromosomes, is the formation of chromosomal multimers. These multimers must be resolved through the use of Xer recombinases at a site called Dif. Without this, daughter cells cannot be segregated<ref>PMID: 21223577</ref>.

Discussion on the importance of the Dif site intensified after a study by Hendrickson and Lawrence (2007) suggested that it was the main site of replication termination <ref>PMID: 17376071</ref>. Prior to this, work suggested that numerous sites called Ter sites were the key to termination. These sites were arranged in a fashion such that they would “trap” and “arrest” termination in the terminus region <ref>PMID:10209736</ref>. However, Hendrickson and Lawrences' (2007) study utilized bioinformatics to suggest an alternative site of termination – the dif site. This inference was made by analysing G-C skew along the replication terminus. GC skew occurs because the lagging strand is more prone to mutation. Their cumulative plot of GC skewing demonstrated a peak where fork arrest occurred. The work by Hendrickson and Lawrence discovered such a peak very close to the dif site but not near any Ter site <ref>PMID: 17376071</ref>. Indeed, this finding held true in 8 different bacteria studied. While they did not directly suggest that the dif site was where termination occurred, they proposed a secondary mechanism whereby XerC/D recombinases binding to the dif site blocked the replication fork <ref>PMID: 17376071</ref>.

Duggin and Bell (2009) challenged the earlier findings and addressed these by utilizing 2D agarose gels to examine cells undergoing different stages of replication. Their findings showed clearly that there was fork arrest at ter sites and that these occurred at a reliable frequency that depended on their natural efficiency to induce fork arrest <ref>PMID: 19233209</ref>. Furthermore when they studied dif sites using a set of fragments of the dif region they found that there was no significant fork arrest anywhere near the dif region. Using these results they conclusively showed that the fork trap model with Ter sites was the most likely model of replication termination.

While the work performed in previous studies had studied bacteria, Duggin and Bell (2011) most recently performed a study on the Archaeon ''Sulfolobus solfataricus''. This Archea shares a degree of similiarity with the previously studied bacteria in possessing a circular chromosome with replication origins and also possessing dif sites. Importantly however, the paper showed that the dif site is not implicated in termination and furthermore exists outside fork fusion zones <ref>PMID: 21113132</ref>.

== Evolution of Tus and RTP systems ==

The study of Tus and the RTP systems reveal two systems which are key in the termination of replication in their hosts. However, while both systems induce termination, considerable differences in their structure and mechanisms of action strongly indicate that the systems evolved independently <ref>PMID:8857533</ref>.

Studies of the Tus-ter system have revealed the evolutionary development of the Tus protein in ''E. Coli'' <ref>PMID: 11523786</ref>. Although it has not been conserved identically in other species, there are organisms in which Tus-like proteins with high sequence homology have been identified <ref>PMID: 16148308</ref>. A study by Rocklein and Kuempel (1992) specifically identified ''S. enterica'' serovar Typhimurium as carrying a termination protein very similar to Tus with 78% sequence homology <ref>PMID: 1495392</ref>. More recent work has identified Tus-ter systems in ''Y. pestis'' and ''K. pneumonia'' both with sequence homologies over 50% <ref>PMID: 11523786</ref>. Furthermore, there have been studies showing Tus-like genes which have been found in plasmids. Specifically two proteins R394 and rts-1 have been found by Koch in two IncT plasmids <ref>PMID: 11106794</ref>.

Likewise, the RTP system of ''B. subtilius'' has many homologues in other species. Griffiths ''et al.'', (1998) found RTP homologs in ''B.atrophaeus'', ''b. amyloliquefaciens'', ''B mojavensis'' and ''B. vallismortis''. The sequences of RTP across the species were similar, but had up to 17.2% (''B. atrophaeus'') different bases. Yet despite this, only five amino acids were changed <ref>PMID: 9642188</ref>. This contrasts with Tus evolution, which has shown much greater sequence differences.

The research on the development of RTP in Bacillus utilized phylogenetic trees mapping the different species <ref>PMID: 11058132</ref>. This allowed the study to conclude that the RTP gene arose only after they had diverged from a common ancestor known as ''B. licheniformis'' <ref>PMID: 9642188</ref>. A more recent study, mapped the entire genome of ''B. licheniformis'' confirming that no RTP like gene existed. Furthermore, no RTP gene has been found on ''B. Halodurans'', narrowing the window in which RTP likely arose <ref>PMID: 15461803</ref>.

== References ==
<references/>

Alexei Ilinykh: uploaded a new version of "Image:Tus secondary structure.jpg"

2011-05-22T01:21:54Z

Alexei Ilinykh:

== Termination sites in ''B.subtilis'' and ''E. coli'' ==
[[Image:EColi_chromosome_map.jpg|thumb|"border"|||300x300px|||Chromosome map of the 4.6 Mb ''E.Coli'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown.]]

Many bacteria that have a circular chromosome utilise a bidirectional mechanism of DNA replication (Messer, 2002). Replication proceeds from the ''OriC'' site in two directions and terminate at some point ~180° away. DNA replication in Gram-negative ''Escherichia coli'' and Gram-positive ''Bacillus subtilis'' terminates at discrete Ter sites. Ter sites block the movement of the replication fork when the replication fork approaches from one direction (the non-permissive direction) but not the other (permissive direction). Ter sites stall replication forks only when bound by proteins Tus in ''E.Coli'' and Replication Termination Protein (RTP) in ''B.subtilis'' (Hill, 1992). This molecular “trap” restricts the meeting of the replication forks to a small region of the chromosome and may be necessary for orderly segregation of the daughter strands (Duggin et al., 2008).

=== Ter sites in E.Coli ===

There are 10 Ter sites present in ''E.Coli'' (Griffiths et al., 1998). ''TerC'', ''TerB'', ''TerF'', ''TerG'' and ''TerJ'' are oriented to block the clockwise movement of replication forks whilst ''TerA'', ''TerD'', ''TerE'', ''TerI'' and ''TerH'' block the anticlockwise movement of replication forks. Most of the replication forks are trapped in the region between ''TerC'' and ''TerA'' (Breier et al., 2005). If the clockwise replication fork is delayed during DNA replication, termination occurs at ''TerA'' site. If the anticlockwise replication fork is delayed during DNA replication, termination occurs at ''TerC'' site.

Ter sites in E.Coli are 23 bp in length and are asymmetric. A comparison of all Ter sites revealed a consensus sequence (shown below) consisting of a highly conserved 13-bp core region and a strictly conserved GC6 base pair (Kaplan and Bastia, 2009). In the orientation shown, replication forks are blocked when travelling from the left and are unimpeded from the right. The nucleotides that interact with the Tus protein are shown in bold (Neylon et al., 2005)

[[Image:Ter_consensus_sequence.jpg||||"middle"|500x75px|||]]

=== Ter sites in ''B.subtilis'' ===
[[Image:Bsubtilis_chromosome_map.jpg|thumb|"border"|||500x330px|||Chromosome map of the 4.2 Mb ''B. Subtilis'' genome indicating the location and orientation of Ter sites. The two groups of Ter sites are depicted in red and black respectively. An origin of replication (''OriC'') is also shown]]

There are nine Ter sites present in ''B.subtilis'' (Coskun-Ari and Hill, 1997). ''TerIX'', ''TerV'', ''TerIII'' and ''TerI'' are oriented to block the clockwise movement of replication forks whilst ''TerII'', ''TerVIII'', TerIV, ''TerVII'' and ''TerVI'' block the anticlockwise movement of replication forks. In ''B.subtilis'', replication termination occurs mostly at ''TerI'' site because the anticlockwise replication fork takes longer to reach the termination region.

The ''TerI'' site is ~30 bp in length and consists of two overlapping half-sites: the core and auxiliary sequences (Lewis et al., 1990). The core sequence binds to RTP and this sequence-specific protein-DNA interaction allows the binding of a second RTP protein to the auxiliary sequence (Carrigan et al., 1991). The auxiliary sequence cannot bind to RTP in the absence of the core site. The ''TerI'' site is shown below with nucleotides involved in protein interactions highlighted in bold. In the orientation shown, replication forks are blocked when travelling from the right and are unimpeded from the left.

[[Image:Ter1_site.jpg||||"middle"|500x300px|||]]

=== Comparison of Ter sites in ''E.coli'' and ''B.subtilis'' ===

Both ''E.coli'' and ''B.subtilis'' have a similar number of Ter sites. However, the Ter sites do not show any sequence similarity (Hill, 1992). The Ter sites in ''E.coli'' cover a large proportion of the genome with some sites closer to the origin of replication than the site of strand segregation. In contrast, the Ter sites in "B.subtilis" are clustered together in a small region of the chromosome close to the site of strand segregation.

== Structures of the Replication Termination Proteins ==

=== RTP ===
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah

=== Tus ===
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah
Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah Blah blah blah

== Mechanisms of Polar Arrest ==

=== RTP ===
<Structure load='2EFW' size='500' frame='true' align='right' caption='Structure of RTP complexed with a native RTP B site' scene='Insert optional scene name here' />

The ability of RTP to induce polar fork arrest in ''B. subtilis'' poses a problem, namely, how does a symmetrical dimer block the replication fork? Early analysis of the ''B. subtilis'' ter sites revealed two RTP binding sites, a high affinity core site (B site) and a low affinity auxiliary site (A site)<ref name = "terofDNArepchroandplas">PMID: 10209736</ref> . Mutagenic studies noted that generally contrahelicase activity was inversely correlated with binding affinity, though some mutant forms were also found that showed no loss in DNA binding affinity despite a partial or complete loss in contrahelicase activity <ref name = "terofDNArepchroandplas" /> <ref>PMID: 8670817</ref>.

However, initial studies of RTP suggested that polar fork arrest was more complex than just DNA binding affinity. Separate mutagenesis studies showed that contrahelicase activity was dependent on dimer-dimer interactions between RTP molecules bound to the ter site <ref>PMID: 8622923</ref>. Additionally, early studies of the protein structure predicted an asymmetrical complex would aid in the contrahelicase activity of RTP <ref>PMID: 7867072</ref>.

The issue of RTP and polar fork arrest was further complicated by Duggin ''et al.'' (2001) <ref>PMID: 11224562</ref>. Using X-ray crystallography and NMR spectroscopy, the group found RTP formed a symmetric complex with a synthetic symmetric RTP B site (sRB). It was found that in the symmetrical form, extensive dimer interactions led to an increase in DNA binding affinity. This led to the Differential Binding Affinity (DBA) model of RTP where polar fork arrest activity was generated from binding differences between the high affinity B site and low affinity A site within the ter sequence, due to the differences in contact bases between the two sites.

Soon after the publishment of Duggin ''et al''. (2001) however, a series of experiments displayed the DBA model to be inadequate in explaining the polar fork arrest. Dixon ''et al''. (2005) showed sRB to be a poor terminator sequence ''in vitro'', and found no correlation between fork arrest activity and the affinity of the proximal binding site <ref>PMID: 15657033</ref>. Furthermore, Duggin (2006) showed that fusing peptides to the C-terminus of RTP could eliminate fork arrest activity, independent of size of amino acid sequence. The fusion of the peptide did not affect any DNA binding sites, but rather an area of RTP that was proposed to contact the oncoming helicase <ref>PMID: 16822523</ref>.

Such lines of evidence led to a revaluation of the RTP-ter structure. Porter ''et al''. (2007) determined the crystal structure of RTP with a native RTP B binding site (nRB) <ref>PMID: 17521668</ref>. The paper found that the RTP dimer formed an asymmetric complex with nRB due to differences in downstream base contacts with the α3 recognition helix of RTP, creating a “wing-up”/”wing-down” dimer. Modelling of the structure showed the asymmetric complex would allow for extensive protein interactions between the RTP dimers, thus underlying the cooperative binding that is crucial for fork arrest.
Taken together, these lines of evidence suggest that the polar fork arrest activity of RTP is dependent upon asymmetric contacts between RTP and the oncoming helicase, as mediated by the asymmetric interactions with the C-terminus <ref name = "mechofpolforkarr">PMID: 19298368</ref>.

===Tus===
<Structure load='2EWJ' size='500' frame='true' align='right' caption='Structure of the Tus-''Ter''B complex' scene='Insert optional scene name here' />

Despite the polar fork arrest activity of the Tus-ter complex in E. coli, fundamental differences exist between it and the RTP-ter system found in ''B. subtilis''. A most basic difference is the fact Tus binds to ter as a monomer, rather than a dimer as is the case with RTP, and that this intrinsic asymmetry was likely to be the basis of Tus’ polar fork arrest activity <ref name = "mechofpolforkarr" />. Furthermore, the crystal structures of RTP and Tus show vastly different tertiary protein folds and DNA binding motifs <ref name = "strofrepterprowithdna">PMID: 8857533</ref>.

Early studies of the Tus fork arrest mechanism led to the formation of two separate models; the “clamp model” and the “interactions model” <ref name = "repterinecoli">PMID: 16148308</ref>. Structural analysis appeared to support the “clamp model”, showing Tus would bind tightly to DNA and impede sterically the replication fork with the αIV and αV helices <ref name = "strofrepterprowithdna" />. However, some biochemical data seemed to indicate that a strong interaction occurred between Tus and the helicase. For instance, Bastia et al. (2001) showed that protein-protein interactions existed between Tus and DnaB using GST affinity chromatography and the yeast two-hybrid system. Furthermore, a series of mutants were identified with reduced Tus-DnaB interactions. These mutants were found to have substitutions within the L1 loop, and were shown to be deficient in fork arrest activity despite having higher DNA binding affinities <ref>PMID: 11493686</ref>. However, much of the biochemical data is inconsistent, and Tus has been known to block fork arrest of unrelated helicases in a polar manner, both supporting the clamp model <ref name = "repterinecoli" />.

Cross ''et al''. (2006) presented evidence for a modified clamp model <ref>PMID: 16814717</ref>. Using a series of forked Tus-ter complexes, the group was able to show that fork progression from the non-permissive face leads to an increase in the stability of the complex. Further analysis of the forked complexes identified the C (6) residue in ter to be responsible for the locking via a base flipping mechanism. X-ray crystallography found the cytosine residue flipped in a binding pocket where it could hydrogen bond to Gly 149, His 144 and Leu 150 as well as forming hydrophobic interactions with Ile 79 and Phe 140. This “molecular mousetrap” was proposed to be the basis of fork arrest on the non-permissive face of Tus. Doubly forked complexes showed Tus was removed from the complex as DNA contacts were removed as the fork progressed on the permissive side, thus accounting for polarity.

However, recent evidence suggests specific interactions between Tus and the oncoming helicase may be the basis of polar fork arrest. Using a sliding helicase lacking strand separation activity, Bastia ''et al''. (2008) showed Tus was able to block the sliding helicase on the non-permissive side even when the bases preceding C(6) were cross linked, and when the cytosine had been substituted. Hence, Tus was able to induce fork arrest without the base flipping mechanism. Furthermore, the group showed mutations in the L1 loop of Tus diminished its ability to arrest the sliding helicase <ref>PMID: 18708526</ref>.

Such evidence led to the establishment of a model for polar fork arrest as seen in Bastia and Kaplan (2009) <ref name = "mechofpolforkarr" />. In this mechanism, a helicase approaching from the non-permissive face of the Tus-ter complex specifically interacts with Tus, whilst also being sterically impeded. In this mechanism, the base-flipping lock is used a fail-safe to ensure fork arrest. At the same time, a helicase approaching from the permissive face would be able to dislodge Tus due to the lack of protein-protein interactions.

=== Comparison between the mechanism of RTP and Tus ===
It is important to note that there is a degree of similarity between the polar arrest mechanism of RTP and Tus, in so far that specific interactions with an oncoming helicase is required for its impediment. In both cases, the polarity is established by a structural asymmetry. The idea that the two proteins have some basic mechanistic similarity is supported by the fact that RTP can arrest fork progression in ''E. coli'', as can Tus in ''B. subtilis'', albeit with far less efficiency <ref name = "mechofpolforkarr" />.

However, this asymmetry is accomplished in distinct ways; Tus is an inherently asymmetric monomer, whereas RTP takes on an asymmetric conformation once bound to DNA. RTP also lacks any specific base locking mechanism, as seen by the flipping of C (6) in the Tus-ter complex. The fact that such different proteins can have similar functions whilst sharing some basic similarities in mechanism supports the idea of independent, convergent evolution of the systems in ''E. coli'' and ''B. subtilis'' <ref name = "strofrepterprowithdna" />.

== The Dif site: The real termination point? ==

In bacteria, replication of DNA occurs by the way of two replication forks diverging away from the origin of replication, ''oriC'', before fusing and terminating at the terminus region. This system of replication is utilized in both ''E. Coli'' and ''B. Subtilis'' <ref>PMID: 10931283</ref>. These prokaryotes utilize two distinct termination systems, Tus and RTP respectively, to end replication. One interesting feature of replication in organisms with circular chromosomes, is the formation of chromosomal multimers. These multimers must be resolved through the use of Xer recombinases at a site called Dif. Without this, daughter cells cannot be segregated<ref>PMID: 21223577</ref>.

Discussion on the importance of the Dif site intensified after a study by Hendrickson and Lawrence (2007) suggested that it was the main site of replication termination <ref>PMID: 17376071</ref>. Prior to this, work suggested that numerous sites called Ter sites were the key to termination. These sites were arranged in a fashion such that they would “trap” and “arrest” termination in the terminus region <ref>PMID:10209736</ref>. However, Hendrickson and Lawrences' (2007) study utilized bioinformatics to suggest an alternative site of termination – the dif site. This inference was made by analysing G-C skew along the replication terminus. GC skew occurs because the lagging strand is more prone to mutation. Their cumulative plot of GC skewing demonstrated a peak where fork arrest occurred. The work by Hendrickson and Lawrence discovered such a peak very close to the dif site but not near any Ter site <ref>PMID: 17376071</ref>. Indeed, this finding held true in 8 different bacteria studied. While they did not directly suggest that the dif site was where termination occurred, they proposed a secondary mechanism whereby XerC/D recombinases binding to the dif site blocked the replication fork <ref>PMID: 17376071</ref>.

Duggin and Bell (2009) challenged the earlier findings and addressed these by utilizing 2D agarose gels to examine cells undergoing different stages of replication. Their findings showed clearly that there was fork arrest at ter sites and that these occurred at a reliable frequency that depended on their natural efficiency to induce fork arrest <ref>PMID: 19233209</ref>. Furthermore when they studied dif sites using a set of fragments of the dif region they found that there was no significant fork arrest anywhere near the dif region. Using these results they conclusively showed that the fork trap model with Ter sites was the most likely model of replication termination.

While the work performed in previous studies had studied bacteria, Duggin and Bell (2011) most recently performed a study on the Archaeon ''Sulfolobus solfataricus''. This Archea shares a degree of similiarity with the previously studied bacteria in possessing a circular chromosome with replication origins and also possessing dif sites. Importantly however, the paper showed that the dif site is not implicated in termination and furthermore exists outside fork fusion zones <ref>PMID: 21113132</ref>.

== Evolution of Tus and RTP systems ==

The study of Tus and the RTP systems reveal two systems which are key in the termination of replication in their hosts. However, while both systems induce termination, considerable differences in their structure and mechanisms of action strongly indicate that the systems evolved independently <ref>PMID:8857533</ref>.

Studies of the Tus-ter system have revealed the evolutionary development of the Tus protein in ''E. Coli'' <ref>PMID: 11523786</ref>. Although it has not been conserved identically in other species, there are organisms in which Tus-like proteins with high sequence homology have been identified <ref>PMID: 16148308</ref>. A study by Rocklein and Kuempel (1992) specifically identified ''S. enterica'' serovar Typhimurium as carrying a termination protein very similar to Tus with 78% sequence homology <ref>PMID: 1495392</ref>. More recent work has identified Tus-ter systems in ''Y. pestis'' and ''K. pneumonia'' both with sequence homologies over 50% <ref>PMID: 11523786</ref>. Furthermore, there have been studies showing Tus-like genes which have been found in plasmids. Specifically two proteins R394 and rts-1 have been found by Koch in two IncT plasmids <ref>PMID: 11106794</ref>.

Likewise, the RTP system of ''B. subtilius'' has many homologues in other species. Griffiths ''et al.'', (1998) found RTP homologs in ''B.atrophaeus'', ''b. amyloliquefaciens'', ''B mojavensis'' and ''B. vallismortis''. The sequences of RTP across the species were similar, but had up to 17.2% (''B. atrophaeus'') different bases. Yet despite this, only five amino acids were changed <ref>PMID: 9642188</ref>. This contrasts with Tus evolution, which has shown much greater sequence differences.

The research on the development of RTP in Bacillus utilized phylogenetic trees mapping the different species <ref>PMID: 11058132</ref>. This allowed the study to conclude that the RTP gene arose only after they had diverged from a common ancestor known as ''B. licheniformis'' <ref>PMID: 9642188</ref>. A more recent study, mapped the entire genome of ''B. licheniformis'' confirming that no RTP like gene existed. Furthermore, no RTP gene has been found on ''B. Halodurans'', narrowing the window in which RTP likely arose <ref>PMID: 15461803</ref>.

== References ==
<references/>