Bacterial Replication Termination: Difference between revisions
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<StructureSection load=' | <StructureSection load='2ewj' size='350' side='right' scene='Bacterial_Replication_Termination/Tus_opening/1' caption='E. coli TER-binding protein complex with DNA and I- ion (purple) (PDB code [[2ewj]])'> | ||
In most bacterial DNA replication, initiation occurs at an origin where, due to the circular nature of the chromosome, the replication forks move bidirectionally to end at approximately 180 degrees away, at a specific sequence termini region [1]. Bacterial replication termination systems have been well studied in ''Eschericia coli'' and ''Bascillus subtilis''. In both systems a ''trans''-acting replication termination protein binds to a specific ''cis''-acting DNA sequences; the replication termini (''ter''), and the DNA-protein complex arrests the progression of replication forks [2]. The terminator sites are orientated so that protein binding is asymmetric, allowing the complexes to block the replication machinery from only one direction while letting them proceed unimpeded from the other direction [1]. In this way they are said to act in a polar manner. The proteins involved in this termination are non-homologous and differ structurally in ''E.coli'' and ''B.subtilis'', although each contains similar contrahelicase activity and performs similar functions in arresting replication [1]. | In most bacterial DNA replication, initiation occurs at an origin where, due to the circular nature of the chromosome, the replication forks move bidirectionally to end at approximately 180 degrees away, at a specific sequence termini region [1]. Bacterial replication termination systems have been well studied in ''Eschericia coli'' and ''Bascillus subtilis''. In both systems a ''trans''-acting replication termination protein binds to a specific ''cis''-acting DNA sequences; the replication termini (''ter''), and the DNA-protein complex arrests the progression of replication forks [2]. The terminator sites are orientated so that protein binding is asymmetric, allowing the complexes to block the replication machinery from only one direction while letting them proceed unimpeded from the other direction [1]. In this way they are said to act in a polar manner. The proteins involved in this termination are non-homologous and differ structurally in ''E.coli'' and ''B.subtilis'', although each contains similar contrahelicase activity and performs similar functions in arresting replication [1]. | ||
[[Image:Bidirectionalrep2.jpg | thumb | | [[Image:Bidirectionalrep2.jpg | thumb | left | 500px | Bacterial replication fork [3]]] | ||
{{Clear}} | {{Clear}} | ||
==Termination (''ter'') Sites== | ==Termination (''ter'') Sites== | ||
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==Replication Terminator Protein (''Bacillus subtilis'')== | ==Replication Terminator Protein (''Bacillus subtilis'')== | ||
<scene name='User:Bianca_Varney/Bacterial_Replication_Termination/Opening_rtp/1'>The Replication Terminator Protein (RTP) complexed to it's ter site</scene> ([[1f4k]]). | |||
Replication Termination Protein (RTP), found in ''Bacillus subtilis'', is a member of the ‘winged helix’ protein family, and terminates bacterial DNA replication by arresting the replication forks through interactions with DNA in a sequence specific manner [7]. RTP blocks the replication fork through contrahelicase activity; the ability to specifically inhibit the helicase replication machinery and has an additional role in arresting transcription [1][2]. In ''B. subtilis'' the bipartite ''ter'' sequence is overlapping, and each inverted repeat contains core (IRIB) and an auxillary (IRIA) sites [1]. RTP binds to these sequences, resulting in the impediment the replication fork helicase. | Replication Termination Protein (RTP), found in ''Bacillus subtilis'', is a member of the ‘winged helix’ protein family, and terminates bacterial DNA replication by arresting the replication forks through interactions with DNA in a sequence specific manner [7]. RTP blocks the replication fork through contrahelicase activity; the ability to specifically inhibit the helicase replication machinery and has an additional role in arresting transcription [1][2]. In ''B. subtilis'' the bipartite ''ter'' sequence is overlapping, and each inverted repeat contains core (IRIB) and an auxillary (IRIA) sites [1]. RTP binds to these sequences, resulting in the impediment the replication fork helicase. | ||
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====RTP Mechanism of Action==== | ====RTP Mechanism of Action==== | ||
An RTP dimer binds the core sequence and the complex formed allows a second dimer to cooperatively to bind to the auxiliary site [5]. In the absence of a core site, the auxiliary site is unable to bind RTP [5]. Furthermore, without the auxiliary site, RTP is unable to block the replication fork, as the interaction of both dimers has been suggested to provide enough DNA binding strength to displace the replication fork [5]. This binding explains how the symmetrical RTP can block replication helicase machinery in an asymmetric manner. The blocking end occurs at the core site, while it is believed that the non-blocking auxiliary site may let replication through as there is less contact points of the dimer to the DNA and the replication machinery coming from this direction is predicted to displace the dimer that is weakly bound to the auxiliary site, which would then displace the dimer bound to the core [5]. Biochemical and mutational studies have identified particular residues that are vital for the functionality of RTP. Mutations within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability [1]. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically [1]. | An RTP dimer binds the core sequence and the complex formed allows a second dimer to cooperatively to bind to the auxiliary site [5]. In the absence of a core site, the auxiliary site is unable to bind RTP [5]. Furthermore, without the auxiliary site, RTP is unable to block the replication fork, as the interaction of both dimers has been suggested to provide enough DNA binding strength to displace the replication fork [5]. This binding explains how the symmetrical RTP can block replication helicase machinery in an asymmetric manner. The blocking end occurs at the core site, while it is believed that the non-blocking auxiliary site may let replication through as there is less contact points of the dimer to the DNA and the replication machinery coming from this direction is predicted to displace the dimer that is weakly bound to the auxiliary site, which would then displace the dimer bound to the core [5]. Biochemical and mutational studies have identified particular residues that are vital for the functionality of RTP. Mutations within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability [1]. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically [1]. | ||
==The Terminus Utilization Substance (''Escherichia coli'' )== | ==The Terminus Utilization Substance (''Escherichia coli'' )== | ||
<scene name='Bacterial_Replication_Termination/Tus_opening/1'>Tus complexed to the | <scene name='Bacterial_Replication_Termination/Tus_opening/1'>Tus complexed to the E. coli ter site and iodide ions</scene> ([[2ewj]]). | ||
The ''E.coli'' protein that is responsible for termination is a 36kDa protein named Tus (Terminius Utilization Substance) that binds 23bp ''ter'' sites and arrests the replication helicase, DnaB, responsible for separating the two strands of DNA [4][9]. Unlike RTP termination sites, the ten ''E.coli'' ''ter'' sites do not contain inverted sequences or direct repeats and Tus binds as a monomer to a highly conserved core region of 13bp [10]. The tus-''ter'' complex is known to terminate replication by arresting the replication machinery in a in a polar manner however there is great discrepancy in evidence whether Tus specifically interacts or physically blocks the DnaB helicase to arrest its progression. | The ''E.coli'' protein that is responsible for termination is a 36kDa protein named Tus (Terminius Utilization Substance) that binds 23bp ''ter'' sites and arrests the replication helicase, DnaB, responsible for separating the two strands of DNA [4][9]. Unlike RTP termination sites, the ten ''E.coli'' ''ter'' sites do not contain inverted sequences or direct repeats and Tus binds as a monomer to a highly conserved core region of 13bp [10]. The tus-''ter'' complex is known to terminate replication by arresting the replication machinery in a in a polar manner however there is great discrepancy in evidence whether Tus specifically interacts or physically blocks the DnaB helicase to arrest its progression. | ||
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Interestingly, RTP has been found to arrest replication in ''E.coli'' when bound to ''E. coli'' specific''ter'' sequences. This suggests that the Tus-''ter'' complex provides a physical barrier that is not specific to the replication fork [12]. However there is some evidence to suggest that RTP specifically recognizes the ''E.coli''DnaB helicase allowing it to functionally block replicative progression, and that Tus may act similarly [14]. Mutational analysis a contrahelicase region has shown that mutations within these regions abolish RTP's ability to arrest DnaB. This indicates that protein-protein interactions occur between these two proteins, and further structural analysis has identified that these amino acid region interacts with a hinge region on DnB helicase. These reports mean that specific surfaces of the termination proteins, RTP and Tus, could be recognizing the identical or variable surfaces of the helicases [13]. | Interestingly, RTP has been found to arrest replication in ''E.coli'' when bound to ''E. coli'' specific''ter'' sequences. This suggests that the Tus-''ter'' complex provides a physical barrier that is not specific to the replication fork [12]. However there is some evidence to suggest that RTP specifically recognizes the ''E.coli''DnaB helicase allowing it to functionally block replicative progression, and that Tus may act similarly [14]. Mutational analysis a contrahelicase region has shown that mutations within these regions abolish RTP's ability to arrest DnaB. This indicates that protein-protein interactions occur between these two proteins, and further structural analysis has identified that these amino acid region interacts with a hinge region on DnB helicase. These reports mean that specific surfaces of the termination proteins, RTP and Tus, could be recognizing the identical or variable surfaces of the helicases [13]. | ||
==Biological Significance== | ==Biological Significance== | ||
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As most genes are orientated towards the terminus, from the origin, if replication is not arrested, it progresses into regions being actively transcribed and collides into the transcription RNA polymerase [11]. It is also suggested that termination may occur by specific "''dif''" sites; conserved sites that are located near the terminus region that are involved in homologous recombination [15]. In fact the ''dif''-terminus hypothesis proposes that termination occurs at or near these sites, where after termination of the replication forks, the two recombinases, XerC and XerD (proteins originating from ''E.coli''), cause site-specific recombination at these ''dif''-sites, and that this would resolve the concatenated chromosomes and complete replication [15]. This mechanism implies that this replication termination by RTP and Tus proteins is merely advantageous to the bacteria and not necessary [15]. | As most genes are orientated towards the terminus, from the origin, if replication is not arrested, it progresses into regions being actively transcribed and collides into the transcription RNA polymerase [11]. It is also suggested that termination may occur by specific "''dif''" sites; conserved sites that are located near the terminus region that are involved in homologous recombination [15]. In fact the ''dif''-terminus hypothesis proposes that termination occurs at or near these sites, where after termination of the replication forks, the two recombinases, XerC and XerD (proteins originating from ''E.coli''), cause site-specific recombination at these ''dif''-sites, and that this would resolve the concatenated chromosomes and complete replication [15]. This mechanism implies that this replication termination by RTP and Tus proteins is merely advantageous to the bacteria and not necessary [15]. | ||
</StructureSection> | </StructureSection> | ||
==References== | ==References== | ||