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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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lac repressor–operator complexes block replisome movement in vitro. (A) Replication of plasmid templates with no lac operator sequences (pPM308, lanes 1–4) and with 22 tandem lacO sequences (pIK02, lanes 5–8) in the presence of 0, 5, 25 and 100 nM LacI tetramers. Products of replication were analysed on a denaturing agarose gel, with DNA size markers shown in kb. (B) Production of distinct 2 and 4 kb leading strand products on template pIK02 (lacO22) bound by LacI is abrogated by addition of IPTG. LacI (100 nM tetramers) and IPTG (1 mM) were present as indicated.
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fig1: lac repressor–operator complexes block replisome movement in vitro. (A) Replication of plasmid templates with no lac operator sequences (pPM308, lanes 1–4) and with 22 tandem lacO sequences (pIK02, lanes 5–8) in the presence of 0, 5, 25 and 100 nM LacI tetramers. Products of replication were analysed on a denaturing agarose gel, with DNA size markers shown in kb. (B) Production of distinct 2 and 4 kb leading strand products on template pIK02 (lacO22) bound by LacI is abrogated by addition of IPTG. LacI (100 nM tetramers) and IPTG (1 mM) were present as indicated.

Mentions: To assess the ability of the replication apparatus of E.coli to duplicate protein-bound DNA, the impact of lac repressor-operator complexes on replisome movement was analysed in vitro. DnaA-directed bidirectional replication of oriC-containing plasmids resulted in generation of ≈0.5 kb lagging strands plus ≈3 kb leading strands formed by convergence of the two forks in a region opposite oriC (Figure 1A, lane 1) (18). The sizes of leading and lagging strands generated on a plasmid lacking lacO sequences was not altered upon addition of lac repressor (LacI) (Figure 1A, lanes 1–4). Non-sequence-specific binding of LacI was not therefore a major impediment to replisome movement. However, addition of LacI to plasmid encoding 22 lacO sites located 2 and 4 kb from oriC lead to accumulation of leading strands of 2 and 4 kb in size (Figure 1A, lanes 5–8). Addition of the inducer IPTG to reactions with LacI on template DNA containing lacO22 precluded appearance of 2 and 4 kb leading strand products (Figure 1B). These data demonstrate that LacI–lacO complexes inhibit replisome movement, allowing convergence of the two forks at lacO22.


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)

lac repressor–operator complexes block replisome movement in vitro. (A) Replication of plasmid templates with no lac operator sequences (pPM308, lanes 1–4) and with 22 tandem lacO sequences (pIK02, lanes 5–8) in the presence of 0, 5, 25 and 100 nM LacI tetramers. Products of replication were analysed on a denaturing agarose gel, with DNA size markers shown in kb. (B) Production of distinct 2 and 4 kb leading strand products on template pIK02 (lacO22) bound by LacI is abrogated by addition of IPTG. LacI (100 nM tetramers) and IPTG (1 mM) were present as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636447&req=5

fig1: lac repressor–operator complexes block replisome movement in vitro. (A) Replication of plasmid templates with no lac operator sequences (pPM308, lanes 1–4) and with 22 tandem lacO sequences (pIK02, lanes 5–8) in the presence of 0, 5, 25 and 100 nM LacI tetramers. Products of replication were analysed on a denaturing agarose gel, with DNA size markers shown in kb. (B) Production of distinct 2 and 4 kb leading strand products on template pIK02 (lacO22) bound by LacI is abrogated by addition of IPTG. LacI (100 nM tetramers) and IPTG (1 mM) were present as indicated.
Mentions: To assess the ability of the replication apparatus of E.coli to duplicate protein-bound DNA, the impact of lac repressor-operator complexes on replisome movement was analysed in vitro. DnaA-directed bidirectional replication of oriC-containing plasmids resulted in generation of ≈0.5 kb lagging strands plus ≈3 kb leading strands formed by convergence of the two forks in a region opposite oriC (Figure 1A, lane 1) (18). The sizes of leading and lagging strands generated on a plasmid lacking lacO sequences was not altered upon addition of lac repressor (LacI) (Figure 1A, lanes 1–4). Non-sequence-specific binding of LacI was not therefore a major impediment to replisome movement. However, addition of LacI to plasmid encoding 22 lacO sites located 2 and 4 kb from oriC lead to accumulation of leading strands of 2 and 4 kb in size (Figure 1A, lanes 5–8). Addition of the inducer IPTG to reactions with LacI on template DNA containing lacO22 precluded appearance of 2 and 4 kb leading strand products (Figure 1B). These data demonstrate that LacI–lacO complexes inhibit replisome movement, allowing convergence of the two forks at lacO22.

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