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Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.

Payne BT, van Knippenberg IC, Bell H, Filipe SR, Sherratt DJ, McGlynn P - Nucleic Acids Res. (2006)

Bottom Line: However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes.Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks.We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

ABSTRACT
All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein-DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.

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Related in: MedlinePlus

Replication blockage occurs on linearized as well as supercoiled template DNA. (A) In the absence of a topoisomerase, replication can initiate at oriC but is inhibited after about 1 kb of synthesis (i). Addition of SmaI results in cleavage of the DNA near to oriC (ii and iii) and allows one of the two forks to progress around the now-linearized template. Unimpeded replication would generate leading strands of 6 kb for both forks (v and vii) whereas 4 and 2 kb leading strands would result from blockage of the forks at lacO (iv and vi). (B) Replication of pPM308 (lacO0), pPM437 (lacO3), pD506 (lacO6) and pIK02 (lacO22) in the absence of a topoisomerase but with LacI and SmaI as indicated, monitored by denaturing agarose gel electrophoresis. DNA size markers are shown in kb. The position of early replication intermediate (ERI) (37), which accumulated in the absence of SmaI, is shown using pPM308 as template (lane 1).
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fig3: Replication blockage occurs on linearized as well as supercoiled template DNA. (A) In the absence of a topoisomerase, replication can initiate at oriC but is inhibited after about 1 kb of synthesis (i). Addition of SmaI results in cleavage of the DNA near to oriC (ii and iii) and allows one of the two forks to progress around the now-linearized template. Unimpeded replication would generate leading strands of 6 kb for both forks (v and vii) whereas 4 and 2 kb leading strands would result from blockage of the forks at lacO (iv and vi). (B) Replication of pPM308 (lacO0), pPM437 (lacO3), pD506 (lacO6) and pIK02 (lacO22) in the absence of a topoisomerase but with LacI and SmaI as indicated, monitored by denaturing agarose gel electrophoresis. DNA size markers are shown in kb. The position of early replication intermediate (ERI) (37), which accumulated in the absence of SmaI, is shown using pPM308 as template (lane 1).

Mentions: Omission of a topoisomerase from replication reactions still allows initiation of replication at oriC in negatively supercoiled plasmid template (24). However, only one of the two forks initiates DNA synthesis and the fork that does initiate stalls after about 1 kb of DNA synthesis due to accumulation of positive torsional strain (27). This inhibition of fork movement can be relieved by addition of a restriction enzyme (SmaI) that cleaves near oriC thus relieving any topological constraint by linearization of the template (24) [Figure 3A (i–iii)]. Inclusion of radiolabelled dCTP at the time of SmaI addition allows fork progression to be monitored.


Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.

Payne BT, van Knippenberg IC, Bell H, Filipe SR, Sherratt DJ, McGlynn P - Nucleic Acids Res. (2006)

Replication blockage occurs on linearized as well as supercoiled template DNA. (A) In the absence of a topoisomerase, replication can initiate at oriC but is inhibited after about 1 kb of synthesis (i). Addition of SmaI results in cleavage of the DNA near to oriC (ii and iii) and allows one of the two forks to progress around the now-linearized template. Unimpeded replication would generate leading strands of 6 kb for both forks (v and vii) whereas 4 and 2 kb leading strands would result from blockage of the forks at lacO (iv and vi). (B) Replication of pPM308 (lacO0), pPM437 (lacO3), pD506 (lacO6) and pIK02 (lacO22) in the absence of a topoisomerase but with LacI and SmaI as indicated, monitored by denaturing agarose gel electrophoresis. DNA size markers are shown in kb. The position of early replication intermediate (ERI) (37), which accumulated in the absence of SmaI, is shown using pPM308 as template (lane 1).
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Related In: Results  -  Collection

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fig3: Replication blockage occurs on linearized as well as supercoiled template DNA. (A) In the absence of a topoisomerase, replication can initiate at oriC but is inhibited after about 1 kb of synthesis (i). Addition of SmaI results in cleavage of the DNA near to oriC (ii and iii) and allows one of the two forks to progress around the now-linearized template. Unimpeded replication would generate leading strands of 6 kb for both forks (v and vii) whereas 4 and 2 kb leading strands would result from blockage of the forks at lacO (iv and vi). (B) Replication of pPM308 (lacO0), pPM437 (lacO3), pD506 (lacO6) and pIK02 (lacO22) in the absence of a topoisomerase but with LacI and SmaI as indicated, monitored by denaturing agarose gel electrophoresis. DNA size markers are shown in kb. The position of early replication intermediate (ERI) (37), which accumulated in the absence of SmaI, is shown using pPM308 as template (lane 1).
Mentions: Omission of a topoisomerase from replication reactions still allows initiation of replication at oriC in negatively supercoiled plasmid template (24). However, only one of the two forks initiates DNA synthesis and the fork that does initiate stalls after about 1 kb of DNA synthesis due to accumulation of positive torsional strain (27). This inhibition of fork movement can be relieved by addition of a restriction enzyme (SmaI) that cleaves near oriC thus relieving any topological constraint by linearization of the template (24) [Figure 3A (i–iii)]. Inclusion of radiolabelled dCTP at the time of SmaI addition allows fork progression to be monitored.

Bottom Line: However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes.Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks.We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

ABSTRACT
All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein-DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.

Show MeSH
Related in: MedlinePlus