Proteopedia - User contributions [en]

Replication termination in E. coli and B. subtilis

2011-05-23T02:19:04Z

Lauren Fowler: /* References */

==''ter'' elements, Tus and RTP==

Many types of bacteria have a circular chromosome that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the two parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [1]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [2]. These elements are situated in the terminus region, approximately opposite the origin of replication [1]. The binding of specific proteins to ''ter'' elements provides a trap for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [1,3]. The ''ter''-protein complex responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [3,4]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [4,5]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork (Figure 1) is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli [6].

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [7].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [7]. It acts by binding to the ''ter'' site and interfering with the helicase coupled strand separation function of the helicase DnaB in the replication machinery [8]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet (yellow) domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [6]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [7] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permissive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [6].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [7]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein (Figure 4) is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [1]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [6,9]. A 2:1 complex between the RTP dimers and the DNA is therefore formed [3]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [9,10]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [11] and the RTP and the replisome, although this is not well understood [6]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [11].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [11].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [1]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [11]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [6]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [5,10].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used [2].
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].

However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387''':598-603. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T02:04:57Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

Many types of bacteria have a circular chromosome that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the two parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [1]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [2]. These elements are situated in the terminus region, approximately opposite the origin of replication [1]. The binding of specific proteins to ''ter'' elements provides a trap for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [1,3]. The ''ter''-protein complex responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [3,4]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [4,5]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork (Figure 1) is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli [6].

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [7].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [7]. It acts by binding to the ''ter'' site and interfering with the helicase coupled strand separation function of the helicase DnaB in the replication machinery [8]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet (yellow) domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [6]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [7] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permissive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [6].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [7]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein (Figure 4) is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [1]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [6,9]. A 2:1 complex between the RTP dimers and the DNA is therefore formed [3]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [9,10]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [11] and the RTP and the replisome, although this is not well understood [6]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [11].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [11].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [1]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [11]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [6]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [5,10].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used [2].
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].

However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T01:52:59Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

Many types of bacteria have a circular chromosome that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the two parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [1]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [2]. These elements are situated in the terminus region, approximately opposite the origin of replication [1]. The binding of specific proteins to ''ter'' elements provides a trap for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [1,3]. The ''ter''-protein complex responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [3,4]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [4,5]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork (Figure 1) is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli [6].

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [7].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [7]. It acts by binding to the ''ter'' site and interfering with the helicase coupled strand separation function of the helicase DnaB in the replication machinery [8]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [6]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [7] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permissive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [6].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [7]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein (Figure 4) is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [1]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [6,9]. A 2:1 complex between the RTP dimers and the DNA is therefore formed [3]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [9,10]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [11] and the RTP and the replisome, although this is not well understood [6]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [11].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [11].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [1]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [11]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [6]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [5,10].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used [2].
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].

However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T01:52:32Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

Many types of bacteria have a circular chromosome that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the two parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [1]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [2]. These elements are situated in the terminus region, approximately opposite the origin of replication [1]. The binding of specific proteins to ''ter'' elements provides a trap for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [1,3]. The ''ter''-protein complex responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [3,4]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [4,5]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork (Figure 1) is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli [6].

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [7].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [7]. It acts by binding to the ''ter'' site and interfering with the helicase coupled strand separation function of the helicase DnaB in the replication machinery [8]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [6]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [7] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permissive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [6].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [7]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [1]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [6,9]. A 2:1 complex between the RTP dimers and the DNA is therefore formed [3]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [9,10]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [11] and the RTP and the replisome, although this is not well understood [6]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [11].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [11].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [1]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [11]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [6]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [5,10].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used [2].
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].

However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T01:23:13Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the two parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [1]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [2]. These elements are situated in the terminus region, approximately opposite the origin of replication [1]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [1,3]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [3,4]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [4,5]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli [6].

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [7].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [7]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [8]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [6]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [7] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [6].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [7]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [1]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [6,9]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [9,10]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [11] and the RTP and the replisome, although this is not well understood [6]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [11].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [11].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [1]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [11]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [6]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [5,10].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used [2].
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].

However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T01:22:45Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [1]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [2]. These elements are situated in the terminus region, approximately opposite the origin of replication [1]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [1,3]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [3,4]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [4,5]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli [6].

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [7].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [7]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [8]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [6]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [7] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [6].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [7]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [1]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [6,9]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [9,10]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [11] and the RTP and the replisome, although this is not well understood [6]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [11].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [11].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [1]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [11]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [6]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [5,10].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used [2].
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].

However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T01:17:13Z

Lauren Fowler: /* References */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
2. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
5. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
6. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
7. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
8. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
9. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
10. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
11. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
12. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
13. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335.

Replication termination in E. coli and B. subtilis

2011-05-23T01:14:37Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
{{Reflist}}
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
<references/>

Replication termination in E. coli and B. subtilis

2011-05-23T01:13:19Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication <ref>Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210</ref>. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites <ref>2</ref>. These elements are situated in the terminus region, approximately opposite the origin of replication <ref>Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210</ref>. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity <ref>1</ref><ref>3</ref>. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap <ref>3</ref><ref>4</ref>. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium <ref>4</ref><ref>5</ref>. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli.<ref>6</ref>

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
{{Reflist}}
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
<references/>

Replication termination in E. coli and B. subtilis

2011-05-23T01:12:21Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication <ref>1</ref>. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites <ref>2</ref>. These elements are situated in the terminus region, approximately opposite the origin of replication <ref>1</ref>. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity <ref>1</ref><ref>3</ref>. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap <ref>3</ref><ref>4</ref>. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium <ref>4</ref><ref>5</ref>. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli.<ref>6</ref>

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
{{Reflist}}
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
<references/>

Replication termination in E. coli and B. subtilis

2011-05-23T01:09:34Z

Lauren Fowler: /* References */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
{{Reflist}}
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.
<references/>

Replication termination in E. coli and B. subtilis

2011-05-22T22:20:01Z

Lauren Fowler: Undo revision 1247089 by Lauren Fowler (Talk)

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T22:17:05Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication <ref>11</ref>. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites<ref>1</ref>. These elements are situated in the terminus region, approximately opposite the origin of replication <ref>11</ref>.The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity<ref>3</ref><ref>11</ref>. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap<ref>2</ref><ref>3</ref>. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium<ref>2</ref><ref>9</ref>. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli<ref>4</ref>.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T22:14:49Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [<ref>11</ref>]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T22:13:23Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]] 
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

File:DBA model.jpg

2011-05-22T22:12:42Z

Lauren Fowler: The DBA model for a polar RTP-Ter complex, indicating the notation used to specify the various dissociation equilibrium constants of the important reactions. Note that whereas the model shows that RTP binds as a dimer, it does not specify that RTP monomer

The DBA model for a polar RTP-Ter complex, indicating
the notation used to specify the various dissociation equilibrium
constants of the important reactions. Note that whereas
the model shows that RTP binds as a dimer, it does not specify that RTP
monomers cannot assemble cooperatively on the DNA in forming each
dimer-DNA complex (see “Discussion”). In addition, the model does not
specify the structural mechanism giving rise to cooperativity; this could
be mediated through direct protein-protein interactions or by mechanisms
associated with DNA structural changes.
Source: Duggin et al. (2005)

Replication termination in E. coli and B. subtilis

2011-05-22T22:08:58Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene> [11]. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8]. This mechanism of action is believed to involve both asymmetric binding at the ''ter'' site, and protein-protein interactions between the two bound RTP dimers [12] and the RTP and the replisome, although this is not well understood [4]. Two models have been proposed to explain the functional polarity of RTP. The differential binding affinity model (Figure 5) suggests that the different affinities of RTP for the A and B sites are responsible, while the induced conformational change model suggests that polarity comes from RTP forming different conformations when positioned at the different sites [12].

[[Image:DBA_model.jpg]]
'''Figure 5:''' The differential binding affinity model for RTP-''ter'' complex polarity. RTP binds as a dimer and both the A and B sites must be filled to terminate replication, although this only occurs if the replication fork approaches the B site, as shown [12].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T21:50:37Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T21:50:01Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==
.
<Structure load='1bm9' size='300' frame='true' align='right' caption='Figure 4: The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T21:36:02Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. Theses sites have no inverted sequence symmetry or direct repeats and so a 1:1 complex is formed with their cognate binding protein, Tus (Termination utilisation substance) [13].

Tus (Figure 2) is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of the helicase DnaB in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

The mechanism of polar fork arrest by Tus, although not yet completely understood, has been investigated and some important interactions identified [4]. Tus forms asymmetric interactions with ''ter'', due to the DNA sequence and the protein structure, as well as protein-protein interactions with the forefront of the replisome, the helicase DnaB. It was hypothesised by Mulcair ''et al.'' (2006) [13] that when DnaB approaches a Tus-''ter'' complex a structure in DNA that differentially affects dissociation of Tus, depending on the directionality of the replication fork, is produced. They found that production of forked DNA, mimicing the molecular action of DnaB, resulted in high rates of dissociation of Tus if the fork was at the permissive face but tighter binding of the Tus-''ter'' complex if the fork was at the non-permisive face, which was determined to be the result of a single cytosine residue, C(6), entering a binding site on Tus (Figure 3). These results provide a possible mechanism for the directionality of relication fork arrest, although it is clear that other interactions are also involved [4].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T21:02:35Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T21:01:54Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities (Figure 1). 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T16:21:46Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene> [13]. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex Tus dissociates, while at the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T16:20:14Z

Lauren Fowler: /* References */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex, Tus dissociates. At the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113. 
13. Mulcair et al. (2006) A molecular mousetrap determines polarity of termination of DNA replication in E.coli. Cell 125:1309-1319.

Replication termination in E. coli and B. subtilis

2011-05-22T16:17:38Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Figure 2: The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex, Tus dissociates. At the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T16:16:56Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption=''''Figure 2:'''The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]
 '''Figure 3:''' Models illustrating the proposed mechanism of polar fork arrest. Following strand separation by DnaB helicase at the permissive face of the Tus-''ter'' complex, Tus dissociates. At the non-permissive face, Tus becomes locked in place. [13]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T16:12:25Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption=''''Figure 2:'''The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Tus-ter_complex.jpg]]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

File:Tus-ter complex.jpg

2011-05-22T16:11:45Z

Lauren Fowler: Models for dissociation of Tus following DnaB-mediated strand separation at the permissive and non-permissive faces of the Tus-ter complex. Source: Mulcair et al. (2006).

Models for dissociation of Tus following DnaB-mediated strand separation at the permissive and non-permissive faces of the Tus-ter complex.
Source: Mulcair et al. (2006).

Replication termination in E. coli and B. subtilis

2011-05-22T16:08:58Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption=''''Figure 2:'''The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:proteinxxx.jpg]]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T16:08:16Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption=''''Figure 2:'''The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a 36 kDa <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

[[Image:Example.jpg]]

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T16:03:25Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption=''''Figure 2:'''The structure of the Tus protein, bound to DNA.' scene='Insert optional scene name here' />
''E. coli'' possess two clusters of five ''ter'' elements, named ''terA-J'', each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T16:00:31Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]] 
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T16:00:01Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]]
Figure 1: E.coli and B.subtilis replication fork traps. (A) The E.coli dif site, where recombination occurs, and the polar terA-J elements are shown. The fork trap is between the opposing Ter sites. (B) B.subtilis fork trap formed by terI-IX elements. (C) Inner terminus region of E.coli, showing the positions of dif and the tus gene. The arrows illustrate two possible meetings of the replication forks to result in termination of replication. (D) Inner terminus region of B.subtilis, showing the positions of dif and the rtp gene. The ter sites are clustered more towards the centre of the terminus region, compared to in E.coli. [4]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T15:43:48Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:Replication_fork.jpg]]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

File:Replication fork.jpg

2011-05-22T15:42:37Z

Lauren Fowler: The DNA replication fork traps of the E. coli and B. subtilis chromosomes. A. The locations of Ter sites for E. coli (TerA–TerJ) are shown, as is the dif site. The two opposed groups of polar Ter sites form the fork trap; TerC, TerB, TerF, TerG and TerJ

The DNA replication fork traps of the E. coli and B. subtilis chromosomes.
A. The locations of Ter sites for E. coli (TerA–TerJ) are shown, as is the dif site. The two opposed groups of polar Ter sites form the fork trap;
TerC, TerB, TerF, TerG and TerJ are orientated to block only clockwise moving forks, whereas TerA, TerD, TerE, TerI and TerH are orientated
to block only anticlockwise forks. The dashed arrows indicate that DNA strand compositional skew organizes the chromosome into two
segments bisected by the oriC and dif loci (see text).
B. In the B. subtilis fork trap, TerI, TerIII, TerV and TerIX are orientated to block clockwise moving forks, whereas TerII, TerVIII, TerIV, TerVII
and TerVI are orientated to block anticlockwise forks.
C. The inner region of the replication fork trap of E. coli, showing the innermost Ter sites, the position of dif and the location of the tus gene.
The continuous arrows indicate the movement of the first fork to arrive from oriC when the innermost Ter site blocks its movement (TerA or
TerC, depending on whether the anticlockwise or clockwise fork arrives first). The dashed arrows indicate the movement of the second fork to
arrive in each case. Thus, termination would occur at TerA if the clockwise replication fork were delayed, and at TerC if the anticlockwise fork
were delayed. This would occur with reasonable frequency (Breier et al., 2005), but a significant number of forks would meet and fuse within
the region between TerA and TerC.
D. In B. subtilis, the Ter sites are more clustered towards the centre of the terminus region. Note that TerI and TerII are only separated by
~0.1 kb and the location of these two sites is not shown to scale. In B. subtilis 168, DNA replication almost always terminates at TerI because
of the asymmetric location of TerI – the anticlockwise fork has to travel a longer distance than the clockwise fork to reach it. In other strains
of B. subtilis that are cured of the SPb prophage on the anticlockwise replichore, the dif locus would be much closer to 180° from oriC. The
tus (E. coli ) and rtp (B. subtilis) genes are autoregulated by the binding of the terminator proteins to Ter sites within the promoter regions
(Ahn et al., 1993; Natarajan et al., 1991; Roecklein et al., 1991).

Source: Duggin et al. (2008)

Replication termination in E. coli and B. subtilis

2011-05-22T15:37:42Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

[[Image:http://imageshack.us/photo/my-images/171/advproteins.png/]]

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T15:36:53Z

Lauren Fowler: /* ''ter'' elements, Tus and RTP */

==''ter'' elements, Tus and RTP==

The genome of many types of bacteria is circular DNA that is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a trap
for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities. 

<a target='_blank' title='ImageShack - Image And Video Hosting' href='http://imageshack.us/photo/my-images/171/advproteins.png/'><img src='http://img171.imageshack.us/img171/3255/advproteins.png' border='0'/></a>

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T14:07:59Z

Lauren Fowler: /* References */

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' (terminator) elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a "trap" for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.'' '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T14:07:14Z

Lauren Fowler: /* References */

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' (terminator) elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a "trap" for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination structures in the ''Escherichia coli'' chromosome replication fork trap. ''J. Mol. Biol.'' '''387''':532-539. 
2. Kamada ''et al.'' (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian ''et al.'' (2007) An asymmetric structure of the ''Bacillus subtilis'' replication terminator protein in complex with DNA. ''J. Mol. Biol.'' '''370''':481-491. 
4. Duggin ''et al.'' (2008) The replication fork trap and termination of chromosome replication. ''J. Mol. Biol.''. '''70'''(6):1323-1333. 
5. Lemon ''et al.'' (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1):212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6):1867-1882. 
7. Andersen ''et al.'' (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6):1327-1335. 
8. Iiamaa, T and Wake, R. (1987) The normal replication terminus of the ''Bacillus subtilis'' chromosome, ''terC'', is dispensible for vegetative growth and sporulation. ''J. Mol. Biol'' '''195''':299-310. 
9. Roecklein ''et al.'' (1991) The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol.'' '''142''':169-175. 
10. Griffiths ''et al.'' (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11. Wilce ''et al.'' (2001) Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. ''Nature structural biology'' '''8'''(3):206-210. 
12. Duggin et al. (2005) A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J. Biol. Chem. 280(13):13105-13113.

Replication termination in E. coli and B. subtilis

2011-05-22T13:58:20Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. The life cycle of bacteria depends upon the coordinated termination of this DNA replication [11]. 

''ter'' (terminator) elements are asymmetric patterns of DNA that act as protein binding sites [1]. These elements are situated in the terminus region, approximately opposite the origin of replication [11]. The binding of specific proteins to ''ter'' elements provides a "trap" for the proceeding replication fork, catching the replication fork as it passes. There are several ''ter'' elements responsible for stopping each replication fork, with each of these elements being specific for the fork passing in one direction only, that is, they have functional polarity [3,11]. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ''ter'' sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posess two clusters of five ''ter'' elements, named ''ter'' A-J, each of which is 23 base pairs long [3]. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus (Termination utilisation substance) protein.

Tus is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibited strong binding affinity [3].

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''B.subtilis'' posesses nine ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP molecule.

The RTP protein is a 29 kDa member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

It has been found that the ter site DNA in ''E. coli'' and ''B. subtilis'' does not bear any sequence homology [11]. Similarly, the termination proteins Tus and RTP do not share conformational characteristics, three dimensional homology or binding similarities [12]. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al. (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA. 
11.

Replication termination in E. coli and B. subtilis

2011-05-22T12:03:11Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated in the approximate region opposite the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posesses 2 clusters of 23 base pair ''ter'' elements, each with 5 ''ter'' sites named ''ter'' A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus protein.

Tus (Termination Utilisation Substance) is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP, bound to DNA.' scene='Insert optional scene name here' />
''Bacillus subtilis'' posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

The termination proteins Tus and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-22T12:02:44Z

Lauren Fowler: /* B. subtilis and RTP */

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated in the approximate region opposite the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posesses 2 clusters of 23 base pair ''ter'' elements, each with 5 ''ter'' sites named ''ter'' A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus protein.

Tus (Termination Utilisation Substance) is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP.' scene='Insert optional scene name here' />
''Bacillus subtilis'' posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [4,5]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

The termination proteins Tus and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-22T11:58:39Z

Lauren Fowler: /* ''E. coli'' and Tus */

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated in the approximate region opposite the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='The structure of the Tus protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posesses 2 clusters of 23 base pair ''ter'' elements, each with 5 ''ter'' sites named ''ter'' A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus protein.

Tus (Termination Utilisation Substance) is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP.' scene='Insert optional scene name here' />
''Bacillus subtilis'' posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [5,4]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

The termination proteins Tus and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-22T11:27:08Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated in the approximate region opposite the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Structure of the TUS protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posesses 2 clusters of 23 base pair ''ter'' elements, each with 5 ''ter'' sites named ''ter'' A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus protein.

Tus (Termination Utilisation Substance) is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP.' scene='Insert optional scene name here' />
''Bacillus subtilis'' posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [5,4]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''==

The termination proteins Tus and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8,9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-''ter''B deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-22T08:58:57Z

Lauren Fowler:

==''ter'' elements, Tus and RTP==

Bacterial DNA is circular and is replicated by two self-sufficient replication forks, progressing in opposite directions. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated in the approximate region opposite the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and Tus==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Structure of the TUS protein, bound to DNA' scene='Insert optional scene name here' />
''E. coli'' posesses 2 clusters of 23 base pair ''ter'' elements, each with 5 ''ter'' sites named ''ter'' A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'' ''ter'' elements is the Tus protein.

Tus (Termination Utilisation Substance) is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ''ter'' site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a positively charged central cleft that can accomodate 13 base pairs of duplex DNA. Tus binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP==

<Structure load='1bm9' size='300' frame='true' align='right' caption='The structure of RTP.' scene='Insert optional scene name here' />
''Bacillus subtilis'' posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. Two RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Even though the protein and its binding at each site is the same, the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur [5,4]. 

As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5,8].

==The effect of knockout of ''ter'' elements or their cognate binding proteins; the real function of ''ter''.==

The termination proteins TUS and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8, 9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with TUS-''ter''B deletions also exhibited increased rates of DNA overproduction. When TUS protein was provided to such cells, this overproduction was corrected, confirming that the absence of TUS (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-22T08:05:22Z

Lauren Fowler: /* ''E. coli'' and TUS protein */

==To do:==
* Can we change the title?
* Introduction/background
* Fix up references (reference all statements throughout, format reference list)
* Add some more info (the last lecture we were given said to put together a story of the discovery, looking at the experiments over time and changes in the theory, as well as addressing a number of questions [red text throughout lecture] about the mechanism of action, the structures of Tus and RTP, mutational data, directional behaviour, etc.)
* Presentation

[Hey Lauren. I have to go home tomorrow morning and I won't be back in Sydney till Sunday afternoon, but while I'm gone I thought I'd read some of the papers I've got printed out sitting here so I can add some actual information! Let me know if there is anything you want me to do - I could make the powerpoint to go with our page? I'm more than happy to do anything where ever I can.]

==''ter'' elements, TUS and RTP==

Bacterial DNA is circular, and so when it is copied in replication, duplication proceeds in 2 directions at 2 self-sufficient replication forks. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated approximately opposite to the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and TUS protein==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Structure of the TUS protein, bound to DNA' scene='Insert optional scene name here' />
''Escherichia coli'' (''E. coli'') posesses 2 clusters of 23 base pair ter elements, each with 5 ter sites named ter A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'''s ''ter'' elements is the TUS protein.

TUS is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ter site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a central cleft which is positively charged, and can accomodate 13 base pairs of duplex DNA. TUS binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP protein==

<Structure load='1bm9' size='300' frame='true' align='right' caption='the RTP protein as it appears bound to DNA.' scene='Insert optional scene name here' />
''Bacillus subtilis'' (''B. subtilis'') posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. 2 RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Fork arrest function is only active when bound to both A and B sites. Even though the protein and its binding to each site is the same,the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur. [5.4]
As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5.8].

==The effect of knockout of ter elements or their cognate binding proteins; the real function of ''ter''.==

The termination proteins TUS and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8, 9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with TUS-''ter''B deletions also exhibited increased rates of DNA overproduction. When TUS protein was provided to such cells, this overproduction was corrected, confirming that the absence of TUS (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-22T08:04:56Z

Lauren Fowler: /* ''E. coli'' and TUS protein */

==To do:==
* Can we change the title?
* Introduction/background
* Fix up references (reference all statements throughout, format reference list)
* Add some more info (the last lecture we were given said to put together a story of the discovery, looking at the experiments over time and changes in the theory, as well as addressing a number of questions [red text throughout lecture] about the mechanism of action, the structures of Tus and RTP, mutational data, directional behaviour, etc.)
* Presentation

[Hey Lauren. I have to go home tomorrow morning and I won't be back in Sydney till Sunday afternoon, but while I'm gone I thought I'd read some of the papers I've got printed out sitting here so I can add some actual information! Let me know if there is anything you want me to do - I could make the powerpoint to go with our page? I'm more than happy to do anything where ever I can.]

==''ter'' elements, TUS and RTP==

Bacterial DNA is circular, and so when it is copied in replication, duplication proceeds in 2 directions at 2 self-sufficient replication forks. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated approximately opposite to the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and TUS protein==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Structure of the TUS protein, bound to DNA' scene='Insert optional scene name here' />
''Escherichia coli'' (''E. coli'') posesses 2 clusters of 23 base pair ter elements, each with 5 ter sites named ter A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'''s ''ter'' elements is the TUS protein.

TUS is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [[[6]]]. This prevents progression of the DNA replication machinery through the ter site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a central cleft which is positively charged, and can accomodate 13 base pairs of duplex DNA. TUS binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP protein==

<Structure load='1bm9' size='300' frame='true' align='right' caption='the RTP protein as it appears bound to DNA.' scene='Insert optional scene name here' />
''Bacillus subtilis'' (''B. subtilis'') posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. 2 RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Fork arrest function is only active when bound to both A and B sites. Even though the protein and its binding to each site is the same,the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur. [5.4]
As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5.8].

==The effect of knockout of ter elements or their cognate binding proteins; the real function of ''ter''.==

The termination proteins TUS and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8, 9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with TUS-''ter''B deletions also exhibited increased rates of DNA overproduction. When TUS protein was provided to such cells, this overproduction was corrected, confirming that the absence of TUS (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-20T13:35:50Z

Lauren Fowler: /* To do: */

Replication termination in E. coli and B. subtilis

2011-05-20T12:35:25Z

Lauren Fowler: /* To do: */

==To do:==
* Can we change the title?
* Introduction/background
* Fix up references (reference all statements throughout, format reference list)
* Add some more info
* Presentation

[Hey Lauren. I have to go home tomorrow morning and I won't be back in Sydney till Sunday afternoon, but while I'm gone I thought I'd read some of the papers I've got printed out sitting here so I can add some actual information! Let me know if there is anything you want me to do - I could make the powerpoint to go with our page? I'm more than happy to do anything where ever I can.]

==''ter'' elements, TUS and RTP==

Bacterial DNA is circular, and so when it is copied in replication, duplication proceeds in 2 directions at 2 self-sufficient replication forks. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated approximately opposite to the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and TUS protein==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Structure of the TUS protein, bound to DNA' scene='Insert optional scene name here' />
''Escherichia coli'' (''E. coli'') posesses 2 clusters of 23 base pair ter elements, each with 5 ter sites named ter A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'''s ''ter'' elements is the TUS protein.

TUS is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ter site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a central cleft which is positively charged, and can accomodate 13 base pairs of duplex DNA. TUS binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP protein==

<Structure load='1bm9' size='300' frame='true' align='right' caption='the RTP protein as it appears bound to DNA.' scene='Insert optional scene name here' />
''Bacillus subtilis'' (''B. subtilis'') posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. 2 RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Fork arrest function is only active when bound to both A and B sites. Even though the protein and its binding to each site is the same,the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur. [5.4]
As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5.8].

==The effect of knockout of ter elements or their cognate binding proteins; the real function of ''ter''.==

The termination proteins TUS and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8, 9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with TUS-''ter''B deletions also exhibited increased rates of DNA overproduction. When TUS protein was provided to such cells, this overproduction was corrected, confirming that the absence of TUS (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.

Replication termination in E. coli and B. subtilis

2011-05-20T12:34:39Z

Lauren Fowler:

==To do:==
* Introduction/background
* Fix up references (reference all statements throughout, format reference list)
* Add some more info
* Presentation

[Hey Lauren. I have to go home tomorrow morning and I won't be back in Sydney till Sunday afternoon, but while I'm gone I thought I'd read some of the papers I've got printed out sitting here so I can add some actual information! Let me know if there is anything you want me to do - I could make the powerpoint to go with our page? I'm more than happy to do anything where ever I can.]

==''ter'' elements, TUS and RTP==

Bacterial DNA is circular, and so when it is copied in replication, duplication proceeds in 2 directions at 2 self-sufficient replication forks. These forks contain several factors, such as a helicase and single stranded-DNA binding proteins, involved in unwinding and maintaining the separation of the 2 parent strands whilst daughter strands are synthesised. 

''ter'' elements are asymmetric patterns of DNA that act as protein binding sites [1]. The binding of specific proteins to these ''ter'' elements provides a trap for the proceeding replication fork, and catches the replication fork as it passes. There are several ''ter'' elements responsible for catching each replication fork, with each of these elements being specific for the fork passing in one direction only. The ''ter'' element responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing ''ter'' element fork trap [2,3]. These elements are situated approximately opposite to the origin of replication. 

Replication fork traps have been identified in multiple species possessing circular chromosomes, including ''Escherichia coli'', ''Bacillus subtilis'' and more recently ''Salmonella'' typhimurium [2,9]. Fork traps prevent over replication of the bacterial chromosome and stall a faster fork in the case that one side of the replication was proceeding faster than the other. The presence of several ''ter'' sites for each replication fork is necessary to ensure that replication termination occurs and indicates a sense of redundancy, supported by the highly conserved nature of ter sites and their highly specific cognate binding capabilities.

==''E. coli'' and TUS protein==

<Structure load='1ecr' size='300' frame='true' align='right' caption='Structure of the TUS protein, bound to DNA' scene='Insert optional scene name here' />
''Escherichia coli'' (''E. coli'') posesses 2 clusters of 23 base pair ter elements, each with 5 ter sites named ter A-E. They are situated around 100kB either side of the termination region. The cognate binding protein to ''E. coli'''s ''ter'' elements is the TUS protein.

TUS is a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_functional_monomer/1'>functional monomer</scene>. It acts by binding to the ''ter'' site and inhibiting the helicase coupled strand separation function of DnaB helicase in the replication machinery [6]. This prevents progression of the DNA replication machinery through the ter site and contributes to replicative arrest. The TUS protein is composed of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Tus_alpha_and_beta/1'>2 alpha helix domains (pink) joined by a central B sheet domain</scene>. This structure forms a central cleft which is positively charged, and can accomodate 13 base pairs of duplex DNA. TUS binds and acts monomerically, and has exhibits strong binding affinity.

==B. subtilis and RTP protein==

<Structure load='1bm9' size='300' frame='true' align='right' caption='the RTP protein as it appears bound to DNA.' scene='Insert optional scene name here' />
''Bacillus subtilis'' (''B. subtilis'') posesses ''ter'' elements of 29 base pair sequences containing 16 base pair imperfectly inverted repeats [3]. These are highly conserved sequences, which create functional A and B sites. The B site represents a more strongly acting binding core site, whilst the A site represents an auxiliary binding domain with lesser affinity for the RTP protein.

The RTP protein is a member of the winged helix family and consists of <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_alpha_and_beta/1'>alpha helix and beta sheet domains.</scene>. RTP acts as a <scene name='Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis/Rtp_homodimer/1'>functional homodimer</scene>. 2 RTP monomers join to form a dimer which binds to either the A or B site of a ''ter'' element. Fork arrest function is only active when bound to both A and B sites. Even though the protein and its binding to each site is the same,the stronger B site is always filled before the A, and both sites must be filled for fork arrest to occur. [5.4]
As in ''E. coli'', the ''B. subtilis'' fork arrest process is mono-directionally selective. If the replication fork arrives at the A site before the B site, it will pass through unhindered, however if the B site is reached before the A site, the fork is arrested and termination ensues [5.8].

==The effect of knockout of ter elements or their cognate binding proteins; the real function of ''ter''.==

The termination proteins TUS and RTP in ''E. coli'' and ''B. subtilis'' do not share conformational characteristics, three dimensional homology or binding similarities. The only similarity identifyable is their shared biological function, which appears to be an adaption to long term survival with a circular chromosome [4]. Several early experiments showed that under laboratory conditions, the knockout of either ''rtp'' or ''tus'' genes (leading to loss of protein synthesis) does not cause an observable phenotype in either ''B. subtilis'' or ''E. coli'' [8, 9].

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. These include:
* Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the ''ter'' element was to inactivate the region, another ''ter'' element further towards the terminus-to-origin direction might be used. [1]
* Multiple ''ter'' sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed. [10]
However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.

The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [4].

More recent studies have showed a highly important and genome wide regulatory role for the ''ter'' sites and their cognate binding proteins. Study of ''E. coli'' shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ''ter'' sites leads to increased levels of DNA overproduction. Furthermore, cells with TUS-''ter''B deletions also exhibited increased rates of DNA overproduction. When TUS protein was provided to such cells, this overproduction was corrected, confirming that the absence of TUS (and not the loss of polA function) was responsible for the DNA overproduction [6]. Similar studies in ''B. subtilis'' show that when mutations are introduced to partitioning genes in combination with mutation to the ''rtp'' gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins ''spo''IIIE and ''rip''X. ''B. subtilis'' studies show that whilst the loss of ''rtp'' does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [5]. These studies suggest a more global role for the the ''ter'' sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

==References==
1. Duggin, I and Bell, S (2009) Termination Structures in the ''Escherichia coli'' Chromosome Replication Fork Trap. ''J. Mol. Biol''. '''387''', 532-539. 
2. Kamada et al. (1996) Structure of a replication-terminator protein complexed with DNA. ''Nature'' '''387'''. 
3. Vivian et al. (2007) An Asymmetric Structure of the ''Bacillus subtilis'' Replication Terminator Protein in Complex with DNA. ''J. Mol. Biol'' '''370'''. 481-491. 
4. Duggin et al. (2008) The replication fork trap and termination of chromosome replication. ''Molecular biology''. '''70'''(6) 1323-1333. 
5. Lemon et. al. (2000) Effects of replication termination mutants on chromosome partitioning in ''Bacillus subtilis''. ''PNAS'' '''98'''(1) 212-217. 
6. Markovitz, A. (2005) A new ''in vivo'' termination function for DNA polymerase I of ''Escherichia coli'' K12. ''Molecular Microbiology'' '''55'''(6) 1867-1882. 
7. Andersen et al (2000) Functional specificity of the replication fork-arrest complexes of ''Bacillus subtilis'' and ''Escherichia coli'': significant specificity for Tus-ter functioning in ''E. coli''. Molecular Microbiology. '''36'''(6) 1327-1335. 
8. Iiamaa, T and Wake, R. (1987). The Normal Replication Terminus of the ''Bacillus subtilis'' Chromosome, ''terC'', is Dispensible for Vegetative Growth and Sporulation. ''J. Mol. Biol''. 195. 299-310. 
9. Roecklein et al. (1991). The ''tus'' gene of ''Escherichia coli'': autoregulation, analysis of flanking sequences and identification of a complementary system in ''Salmonella typhimurium''. ''Res. Microbiol''. '''142'''. 169-175. 
10. Griffiths et al (2008) Introduction to Genetic Analysis. 9th edition. W H Freeman Publishing, USA.