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. 2006;34(18):5194-202.
doi: 10.1093/nar/gkl682. Epub 2006 Sep 25.

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

Bryony T I Payne  1 , Ingeborg C van Knippenberg, Hazel Bell, Sergio R Filipe, David J Sherratt, Peter McGlynn
Affiliations

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

Bryony T I Payne et al. Nucleic Acids Res. 2006.

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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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
Additive blockage of replisome movement by repressor–operator complexes. (A) Replication of plasmid templates bearing 0, 3, 6 and 22 lacO sites (pPM374, 378, 393 and 379) in the presence of 0, 5, 25 and 100 nM LacI tetramers was monitored by denaturing agarose gel electrophoresis. DNA size markers are shown in kb. (B) Replisome blockage as a function of LacI concentration. Open circles, lacO0; filled circles, lacO3; open squares, lacO6; filled triangles, lacO22.
Figure 3
Figure 3
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).
Figure 4
Figure 4
lac repressor–operator complexes present blocks to DNA replication in vivo. (A) Insertion of tandem lac operators within the chromosomal arg locus, linked to an apramycin resistance gene. The direction of replication fork movement is indicated with a dashed arrow. (B) Southern blot of a 1D agarose gel of PvuII digests from strains bearing 0 (BP38), 22 (PM222) and 34 (BP41) lacO repeats together with pPM306, a plasmid bearing an arabinose-inducible lacI gene, both with and without 1 mM IPTG. DNA size markers are shown in kb. The entire apramycin resistance gene was used to probe the blot. The positions of replication intermediates are marked with arrows. (C) Growth curves of strains BP38 (i), PM222 (ii) and BP41 (iii) containing pPM306. Open circles, medium supplemented with glucose (lacI expression repressed); filled circles, medium supplemented with arabinose (lacI expression induced). c.f.u.: colony forming units.
Figure 5
Figure 5
Recombination enzymes are not required in vivo for tolerance of 22 lacO sites bound by repressor. (A) Growth of strains harbouring 22 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). lacO22 strains were PM222 (otherwise wild-type), BP13 (recA), BP22 (recB), BP60 (recF), BP33 (recG), BP19 (ruvABC), BP16 (rep). Individual growth curves are shown but each curve was performed between two and eight times with all curves yielding similar results. C.f.u.: colony forming units. (B) Mean number of cell divisions between 0 and 4 h of growth for strains bearing 22 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).
Figure 6
Figure 6
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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