Resolution of Joint Molecules by RuvABC and RecG Following Cleavage of the Escherichia coli Chromosome by EcoKI

DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions

Expansion of CAG repeats in Escherichia coli is controlled by single-strand DNA exonucleases of both polarities

The expansion of CAG·CTG repeat tracts is responsible for several neurodegenerative diseases, including Huntington disease and myotonic dystrophy. Understanding the molecular mechanism of CAG·CTG repeat tract expansion is therefore important if we are to develop medical interventions limiting expansion rates. Escherichia coli provides a simple and tractable model system to understand the fundamental properties of these DNA sequences, with the potential to suggest pathways that might be conserved in humans or to highlight differences in behavior that could signal the existence of human-specific factors affecting repeat array processing. We have addressed the genetics of CAG·CTG repeat expansion in E. coli and shown that these repeat arrays expand via an orientation-independent mechanism that contrasts with the orientation dependence of CAG·CTG repeat tract contraction. The helicase Rep contributes to the orientation dependence of repeat tract contraction and limits repeat tract expansion in both orientations. However, RuvAB-dependent fork reversal, which occurs in a rep mutant, is not responsible for the observed increase in expansions. The frequency of repeat tract expansion is controlled by both the 5′–3′ exonuclease RecJ and the 3′–5′ exonuclease ExoI, observations that suggest the importance of both 3′and 5′ single-strand ends in the pathway of CAG·CTG repeat tract expansion. We discuss the relevance of our results to two competing models of repeat tract expansion

Illustration of how an intact monomeric circular chromosome might be generated without recombination following DNA double-strand breakage.

<p>In the absence of RecA, DSB repair would be prevented. Instead, a combination of replication fork reversal by RuvABC and DNA degradation by RecBCD could regenerate an intact circular chromosome and promote survival.</p

Sensitivity of recombination defective mutants to EcoKI breaks.

<p>Exponential cultures were treated with 20 µg/ml of 2-AP and relative viability calculated as described in Experimental Procedures. Error bars indicate 95% confidence intervals. (A) Indicated genotypes are in an <i>hsdR⁺ ΔclpX</i> background. The strains used were DL1902 (<i>rec⁺</i>), DL2656 (<i>recA</i>), DL1940 (<i>ΔrecG</i>), DL2659 (<i>ΔrecBCD</i>), DL1938 (<i>ΔruvABC</i>), DL 1962 (<i>ΔrecG ΔruvABC</i>). (B) Indicated genotypes are in an <i>hsdR514 ΔclpX</i> background. The strains used were DL1800 (<i>rec⁺</i>), DL2666 (<i>recA</i>), DL2133 (<i>ΔrecG</i>), DL2675 (<i>ΔrecBCD</i>), DL2114 (<i>ΔruvABC</i>), DL2667 (<i>recA ΔruvABC</i>), DL2671 (<i>recA ΔrecG</i>), DL2676 (<i>ΔrecBCD ΔruvABC</i>), DL2674 (<i>ΔrecBCD ΔrecG</i>), DL2149 (<i>ΔrecG ΔruvABC</i>). (C) Indicated genotypes are in an <i>hsdR⁺ ΔclpX</i> background. The strains used were DL1940 (<i>ΔrecG</i>), DL1938 (<i>ΔruvABC</i>), DL2656 (<i>recA</i>), DL2670 (<i>recA ΔrecG</i>), DL2657 (<i>recA ΔruvABC</i>).</p

Model for the generation of chromosome dimers without Holliday junction cleavage-ligation.

<p>Bacteria, such as <i>E. coli</i>, have circular chromosomes and at normal growth rates reinitiate DNA replication before the previous round of replication has completed and before cell division takes place. This means that an unresolved Holliday junction is a potential barrier to the passage of the next set of replication forks. We propose here that replication through the Holliday junction may be possible and that this may be facilitated by the branch migration protein RecG. A. Chromosome in which a Holliday junction (HJ) has formed following the passage of a replication fork (RF1). A second pair of replication forks (RF2) are shown approaching the Holliday junction. The first two chromosomes to be produced by this replicating structure are labelled C1 and C2. The two chromosomes destined to be made from C1 are labelled C1.1 and C1.2 and the chromosomes destined to be made from C2 are labelled C2.1 and C2.2. The DNA strands that have exchanged to form the Holliday junction (and strands templated on these) are shown in red whereas the DNA strands that have not exchanged (and strands templated on these) are shown in blue. The four double-stranded molecules formed by the second pair of replication forks are shaded in light blue and pink. B. Two new forks (RF2) approaching the Holliday junction. When the pair of RF2 forks approaches the Holliday junction, the positive supercoiling ahead of the forks is predicted to push the junction ahead of them. At some point the forks are likely to stall, presumably because the Holliday junction impedes their progression. At this point, branch migration of the Holliday junction to the fork will lead to a swapping of newly synthesised sister chromosome arms. Chromosomes C1.1 and C2.1 will be connected to the unreplicated arm of C1 while chromosomes C1.2 and C2.2 will be connected to the unreplicated arm of C2. C. Formation of two monomeric and one dimeric chromosome. The replication machinery is reassembled on the two hybrid RF2 forks and replication continues. The figure illustrates the point where the RF2 forks have passed through the Holliday junction leaving the red strands crossed over (CO). The RF1 forks have completed their replication and no longer exist. When the RF2 forks complete replication and meet at the terminus, two monomeic blue chromosomes (C1.1 and C2.2) will have formed as well as one dimeric red (crossover) chromosome (C1.2–C2.1). The shading of the molecules formed by the RF2 replication forks illustrates that the red double strands are crossed over whereas the blue double strands are not.</p

Restriction enzyme sites are underlined and the complementary parts of the primers useful for the crossover strategy are shown in bold.

<p>Restriction enzyme sites are underlined and the complementary parts of the primers useful for the crossover strategy are shown in bold.</p

Pulsed field gel analysis of chromosomal DNA following treatment of Δ<i>clpX</i> mutant strains with 2-AP, prior to cleavage with <i>Not</i>I (A1, A2 and A3) and after cleavage with <i>Not</i>I (B1, B2 and B3).

<p>The <i>S. cerevisiae</i> chromosomes are shown in lane 1 as a molecular size standard, confirming the compression of linear fragments of 450 kb to 1.5 mb into a single band under the conditions used. This band of yeast chromosomes runs at the same position as linearized E.coli DNA (4.5 mb). (A1 and B1) Lanes 2–15, release of linear DNA into pulsed field gels from <i>rec</i>⁺ and recombination defective strains: DL1902, DL2656, DL2659, DL3179, DL3184, DL2600, DL3201, DL3207, DL2670, DL3204, DL3208, DL2673, DL3200, DL3206. (A2 and B2) Lanes 2–15, release of linear DNA into pulsed field gels from <i>rec</i>⁺ and recombination defective strains containing the plasmid pBAD-<i>rusA</i>: DL3122, DL3217, DL3123, DL3218, DL3219, DL3220, DL3221, DL3222, DL3223, DL3224, DL3225, DL3226, DL3227, DL3228. (A3 and B3) Lanes 2–15, release of linear DNA into pulsed field gels from <i>rec</i>⁺ and recombination defective strains containing the plasmid pBAD18: DL3251, DL3252, DL3253, DL3254, DL3255, DL3256, DL3257, DL3258, DL3259, DL3260, DL3261, DL3262, DL3263, DL3264.</p

Effect of <i>xerC</i> and <i>dif</i> mutations on cell sensitivity to EcoKI breaks.

<p>Exponential cultures were treated with 20 µg/ml of 2-AP and relative viability calculated as described in Experimental Procedures. Error bars indicate 95% confidence intervals. In addition to the genotypes shown, all strains carry the Δ<i>clpX</i> deletion. (A) Strains used were DL1902 (<i>rec⁺</i>), DL1930 (<i>rec⁺ xerC</i>), DL2245 (<i>rec⁺ dif</i>), DL1800 (<i>hsdR rec⁺</i>), DL2097 (<i>hsdR rec⁺ xerC</i>) and DL2244 (<i>hsdR rec⁺ dif</i>). (B) Strains used were DL1938 (Δ<i>ruvABC</i>), DL1952 (Δ<i>ruvABC xerC</i>), DL 2249 (Δ<i>ruvABC dif</i>), DL2114 (<i>hsdR ΔruvABC</i>), DL2118 (<i>hsdR ΔruvABC xerC</i>) and DL2248 (<i>hsdR ΔruvABC dif</i>). (C) Strains used were DL1940 (ΔrecG), DL1944 (ΔrecG <i>xerC</i>), DL2346 (Δ<i>recG dif</i>), DL2133 (<i>hsdR ΔrecG</i>), DL2136 (<i>hsdR ΔrecG xerC</i>) and DL2345 (<i>hsdR ΔrecG dif</i>). (D) Strains used were DL2656 (<i>recA</i>), DL2903 (<i>recA dif</i>), DL2666 (<i>hsdR recA</i>) and DL2904 (<i>hsdR recA dif</i>).</p