Rep and PriA helicase activities prevent RecA from provoking unnecessary recombination during replication fork repair

Abstract

The rescue of replication forks stalled on the template DNA was investigated using an assay for synthetic lethality that provides a visual readout of cell viability and permits investigation of why certain mutations are lethal when combined. The results presented show that RecA and other recombination proteins are often engaged during replication because RecA is present and provokes recombination rather than because recombination is necessary. This occurs particularly frequently in cells lacking the helicase activities of Rep and PriA. We propose that these two proteins normally limit the loading of RecA on ssDNA regions exposed on the leading strand template of damaged forks, and do so by unwinding the nascent lagging strand, thus facilitating reannealing of the parental strands. Gap closure followed by loading of the DnaB replicative helicase enables synthesis of the leading strand to continue. Without either activity, RecA loads more frequently on the DNA and drives fork reversal, which creates a chickenfoot structure and a requirement for other recombination proteins to re-establish a viable fork. The assay also reveals that stalled transcription complexes are common impediments to fork progression, and that damaged forks often reverse independently of RecA.[Keywords: DNA repair; RuvABC; RecBCD, RecFOR; Holliday junction] Supplemental material is available at http://www.genesdev.org.

Transcription termination maintains chromosome integrity

“…2). However, RecA is proposed to both promote fork regression and to process regressed forks (8). Consistent with this model, we found that MDS42 lexA3 is hypersensitive to BCM (MIC <25 μg/mL BCM) ( Fig.…”

“…However, they can only rarely result in fork collapses, which are highly damaging to cells (3,(7)(8)(9)11). Our data suggest that Rho removes TEC ahead of replisomes, preventing collision, fork collapse, and DSB formation.…”

“…The approximately twofold increase in DSB formation in MDS42 lexA3 exposed to BCM illustrates the two roles of RecA. RecA promotes fork regression, which sensitizes cells to BCM, but also induces SOS repair of DSBs, which increases BCM-resistance (8). Linear DNA in the Δrep and ΔpriA strains with or without BCM treatment was below the limit of detection.…”

“…DSBs indicate replication fork collapse. They are generated when arrested forks form "chicken foot" structures, which include a Holliday junction (8) (Fig. 1).…”

“…Fork repair is promoted by multiple pathways that involve a variety of DNA repair functions (6). Recombination can restart replisomes blocked by arrested TEC (7,8). Boubakri et al (9) have demonstrated that DNA helicases Rep, DinG, and UvrD resolve head-on collisions between TEC in rrn operons and replisomes.…”