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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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RecA, RecB and RecG are needed in vivo to tolerate 34 lacO sites bound by repressor. (A) Growth of strains harbouring 34 lacO sites and the indicated mutations, plus the lacI plasmid pPM306. Open circles: cells grown in glucose (no lac repressor overexpression); filled circles: cells grown in arabinose (elevated lac repressor); filled triangles: cells grown in arabinose plus IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB), BP44 (recF), BP54 (recG), BP52 (ruvABC), BP47 (rep), BP55 (uvrD). Each curve was performed between two and six times with very similar results. (B) Mean number of cell divisions between 0 and 4 h of growth for strains bearing 34 lacO sites, determined from growth curves as represented in (A). Open bars: cells grown in glucose; filled bars: cells grown in arabinose. Strains are as described in (A). (C) Southern blot of a 1D agarose gel of PvuII digests of chromosomal DNA from strains bearing lacO34, all containing pPM306, grown in the presence of arabinose with and without 1 mM IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB) and BP54 (recG). The entire apramycin resistance cassette was used to generate the radiolabelled probe. Note also that the amounts of chromosomal DNA detected by the probe varied between strains and likely reflected variation between strains in the number of chromosomes per cell. However, the amount of replication intermediate as a proportion of the total DNA signal did not vary greatly (5 to 8%).
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fig6: RecA, RecB and RecG are needed in vivo to tolerate 34 lacO sites bound by repressor. (A) Growth of strains harbouring 34 lacO sites and the indicated mutations, plus the lacI plasmid pPM306. Open circles: cells grown in glucose (no lac repressor overexpression); filled circles: cells grown in arabinose (elevated lac repressor); filled triangles: cells grown in arabinose plus IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB), BP44 (recF), BP54 (recG), BP52 (ruvABC), BP47 (rep), BP55 (uvrD). Each curve was performed between two and six times with very similar results. (B) Mean number of cell divisions between 0 and 4 h of growth for strains bearing 34 lacO sites, determined from growth curves as represented in (A). Open bars: cells grown in glucose; filled bars: cells grown in arabinose. Strains are as described in (A). (C) Southern blot of a 1D agarose gel of PvuII digests of chromosomal DNA from strains bearing lacO34, all containing pPM306, grown in the presence of arabinose with and without 1 mM IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB) and BP54 (recG). The entire apramycin resistance cassette was used to generate the radiolabelled probe. Note also that the amounts of chromosomal DNA detected by the probe varied between strains and likely reflected variation between strains in the number of chromosomes per cell. However, the amount of replication intermediate as a proportion of the total DNA signal did not vary greatly (5 to 8%).

Mentions: Deletion of rep had no major effect on growth with either 22 or 34 lacO sites (Figures 5 and 6). Rep did not therefore have a critical role in promotion of fork movement through these repressor–operator complexes. We also tested whether UvrD, implicated in a fork clearing role (31), aided cell growth in the face of repressor–operator complexes. However, a uvrD mutation had no effect on the ability of cells to grow in the face of lacO34 (Figure 6A and B).


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)

RecA, RecB and RecG are needed in vivo to tolerate 34 lacO sites bound by repressor. (A) Growth of strains harbouring 34 lacO sites and the indicated mutations, plus the lacI plasmid pPM306. Open circles: cells grown in glucose (no lac repressor overexpression); filled circles: cells grown in arabinose (elevated lac repressor); filled triangles: cells grown in arabinose plus IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB), BP44 (recF), BP54 (recG), BP52 (ruvABC), BP47 (rep), BP55 (uvrD). Each curve was performed between two and six times with very similar results. (B) Mean number of cell divisions between 0 and 4 h of growth for strains bearing 34 lacO sites, determined from growth curves as represented in (A). Open bars: cells grown in glucose; filled bars: cells grown in arabinose. Strains are as described in (A). (C) Southern blot of a 1D agarose gel of PvuII digests of chromosomal DNA from strains bearing lacO34, all containing pPM306, grown in the presence of arabinose with and without 1 mM IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB) and BP54 (recG). The entire apramycin resistance cassette was used to generate the radiolabelled probe. Note also that the amounts of chromosomal DNA detected by the probe varied between strains and likely reflected variation between strains in the number of chromosomes per cell. However, the amount of replication intermediate as a proportion of the total DNA signal did not vary greatly (5 to 8%).
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Related In: Results  -  Collection

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fig6: RecA, RecB and RecG are needed in vivo to tolerate 34 lacO sites bound by repressor. (A) Growth of strains harbouring 34 lacO sites and the indicated mutations, plus the lacI plasmid pPM306. Open circles: cells grown in glucose (no lac repressor overexpression); filled circles: cells grown in arabinose (elevated lac repressor); filled triangles: cells grown in arabinose plus IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB), BP44 (recF), BP54 (recG), BP52 (ruvABC), BP47 (rep), BP55 (uvrD). Each curve was performed between two and six times with very similar results. (B) Mean number of cell divisions between 0 and 4 h of growth for strains bearing 34 lacO sites, determined from growth curves as represented in (A). Open bars: cells grown in glucose; filled bars: cells grown in arabinose. Strains are as described in (A). (C) Southern blot of a 1D agarose gel of PvuII digests of chromosomal DNA from strains bearing lacO34, all containing pPM306, grown in the presence of arabinose with and without 1 mM IPTG. Strains were BP41 (otherwise wild-type), BP43 (recA), BP45 (recB) and BP54 (recG). The entire apramycin resistance cassette was used to generate the radiolabelled probe. Note also that the amounts of chromosomal DNA detected by the probe varied between strains and likely reflected variation between strains in the number of chromosomes per cell. However, the amount of replication intermediate as a proportion of the total DNA signal did not vary greatly (5 to 8%).
Mentions: Deletion of rep had no major effect on growth with either 22 or 34 lacO sites (Figures 5 and 6). Rep did not therefore have a critical role in promotion of fork movement through these repressor–operator complexes. We also tested whether UvrD, implicated in a fork clearing role (31), aided cell growth in the face of repressor–operator complexes. However, a uvrD mutation had no effect on the ability of cells to grow in the face of lacO34 (Figure 6A and B).

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