Branch Migration Prevents DNA Loss during Double-Strand Break Repair

The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday) junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements

RecG directs DNA synthesis during double-strand break repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability

25 years on and no end in sight: a perspective on the role of RecG protein.

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced 'adaptive' mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.CJR is supported by a grant from the Biotechnology and Biological Sciences Research Council [BB/K015729/1]