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.

Figure 1.

Synthetic lethality assay. (A) The E. coli replisome complex, reproduced from McGlynn and Lloyd (2002). (Reprinted with permission from Nature Reviews Molecular Cell Biology. ©2002 Macmillan Magazines Ltd. [http://www.nature.com/reviews].) (B) Rescue of a stalled replication fork. Stalling of the fork at a lesion (red rectangle) triggers fork regression. Subsequent processing of the chickenfoot initiated by RecBCD enzyme (panel i) or RuvABC resolvase (panel ii) establishes a fork upstream of the original block. (C) Possible substrates and mechanisms for loading the DnaB hexamer ring on the template for lagging strand synthesis. Green boxes represent RNA primers synthesized by DnaG, and red triangles represent lesions blocking synthesis by Pol III. (D) Structure of pRC7 showing the genetic elements utilized. (E) Segregation of plasmid-free cells in cultures of ΔlacIZYA strain TB28 carrying pRC7 (strain N6027). (F) Synthetic lethality assays showing the effect of priA, rep, and priC on cell viability. All constructs carry a priA⁺ derivative of pRC7 and the chromosomal mutations indicated. The relevant genotype is shown above each photograph, with the strain number in parentheses. The fraction of white colonies is shown below with the number of white colonies/total colonies analyzed in parentheses.

Figure 2.

The viability of priA300 rep cells depends on Holliday junction resolution. (A) Growth and viability of priA300 rep cells. Samples from exponential broth cultures of the strains listed in B grown to A₆₅₀ 0.4 were diluted and plated as described in Materials and Methods, and photographed after 18 h at 37°C. (B) Viable cell numbers (colony-forming units) in exponential-phase broth cultures grown to A₆₅₀ 0.4. (C) Accumulation of fast-growing variants during subculture of priA300 rep cells. A culture of strain N5587 grown in LB broth from a single colony was incubated overnight, diluted 50-fold in fresh broth, and regrown to saturation before streaking a sample on LB agar. The plate was incubated for 18 h. (D) Effect of priA300 and rep on cell morphology. The strains listed in B were grown to A₆₅₀ 0.4 in broth. Merged phase contrast and fluorescence images are shown, displaying Hoechst 33342 fluorescence in cyan. (E) Chronic SOS expression in priA300 rep cells. SOS expression was measured by assaying β-galactosidase activity in exponential cultures of strains carrying sfiA∷lacZ. (F) Effect of priA300, rep, and ruv mutations on sensitivity to UV light. The strains are listed in parentheses next to the genotype. (G) Synthetic lethality assays illustrating the effect of combining priA300, rep, and ruv mutations. ruvA60 is a Tn10 insertion that inactivates ruvA and prevents transcription of the downstream ruvB.

Figure 3.

RecA, RecD, and RecF affect the viability of priA300 rep cells. (A) Synthetic lethality assay illustrating the very low viability of priA300 rep cells lacking ExoV. (B) Recombination without the ExoV activity of RecBCD. The diagram illustrates how recombination initiated at a DNA end via the unwinding activity of RecD-depleted RecBC enzyme (red triangle) might generate an unusual D-loop. (C–F) Synthetic lethality assays illustrating how RecA and RuvABC affect the viability of priA300 rep cells.

Figure 4.

RecA provokes recombination in cells lacking Rep helicase. (A) Synthetic lethality assays showing dependence on RecF and RecA. The lower panel under panel ii shows a white colony streaked on LB (30 h incubation). (B) A model for alternative processing of a stalled fork by Rep and RecA proteins. The diagram depicts how Rep helicase might be directed by SSB protein (green circles) to unwind the lagging strand of a fork with a gap in the leading strand, thus closing the gap. Alternatively, but especially in the absence of Rep, the RecFOR proteins load RecA (blue ovals) on the leading strand template. RecA promotes homologous pairing and strand exchange with the intact sister duplex, driving fork reversal and forming a chickenfoot structure. (C) Possible ways of converting a chickenfoot to a fork (see text for details). (D–G) Synthetic lethality assays demonstrating how the viability of cells lacking Rep helicase is affected by RecA, RecBCD, RuvABC, and RecG.

Figure 5.

Synthetic lethality assays investigating gap closure and DnaB loading in priA300 rep recA strains. (A) Effect of RecQ helicase and RecJ exonuclease. (B) Effect of DNA polymerases II, IV, and V. (C) Requirement for PriC to load DnaB.

Figure 6.

Synthetic lethality assays showing how recombination maintains viability in strains with reduced means to minimize the incidence of stalled RNAP complexes. (A) Requirement for recombination in strains lacking DksA, or GreA and GreB. (B) Holliday junction resolution induces recombination in strains lacking GreA and GreB.

Figure 7.

Interplay between transcription and replication. (A) Models of how RNAP might block replication and provoke recombination. (Panel i) Arrays of stalled or backtracked RNAP complexes (green ovals) accumulate at problematic sites when the cell’s ability to destabilize such complexes is compromised as a result of reduced activity of RNAP modulators (ppGpp, DksA, GreA/B, or Mfd). A replication fork may stall at such an array and reverse to form a chickenfoot, providing ways in which RuvABC, RecA, and RecBCD might restore a viable fork. (Panel ii) With a full complement of RNAP modulators, or with rpo*35 present to reduce the intrinsic stability of RNAP, arrays are less prevalent. However, a fork will often stall for other reasons. Spontaneous dissociation of the leading strand polymerase from the leading strand 3′OH might expose the leading strand template. Provided PriA, or Rep and PriC, are present to close the gap and load DnaB, coupled leading and lagging strand synthesis may resume. Without PriA or Rep, there is greater risk of ssDNA remaining exposed. RecFOR may then load RecA (blue ovals), which may drive fork reversal. Alternatively, PriC may load DnaB in a manner that leaves a gap in the nascent leading strand. (B,C) Synthetic lethality assays illustrating how the viability of cells lacking PriA is affected by a mutation (rpo*35) reducing the intrinsic stability of RNAP complexes and by the presence of RecA and RuvABC proteins.