Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Abstract

Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.

Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2

“…While it is established that Srs2 dismantles Rad51 filaments, it has remained unclear how Srs2 is recruited to the presynaptic complex and other HR intermediates (Antony et al, 2009; Krejci et al, 2003; Marini and Krejci, 2010; Niu and Klein, 2016; Qiu et al, 2013; Sasanuma et al, 2013; Vasianovich et al, 2017; Veaute et al, 2003). In this study, we provide evidence that Srs2 is preferentially recruited to small clusters of RPA that remain embedded within the Rad51 presynaptic complex.…”

“…Highlighting the importance of these enzymes, mutations in human HR helicases that regular HR result in diseases such as Rothmund-Thompson syndrome and Fanconi Anemia, which are characterized by genome instability and increased incidence of cancer (Brosh, 2013). Srs2 is considered a proto-typical antirecombinase due to its well-characterized ability to remove Rad51 from ssDNA (Antony et al, 2009; Krejci et al, 2003; Marini and Krejci, 2010; Niu and Klein, 2016; Qiu et al, 2013; Sasanuma et al, 2013; Vasianovich et al, 2017; Veaute et al, 2003). Deletion of the SRS2 gene results in an increase in recombination frequency, and its importance is further revealed in Δsrs2 Δsgs1 and Δsrs2 ΔRad54 double mutants, which have a synthetic lethal phenotype due the accumulation of toxic recombination intermediates (Ira et al, 2003; Klein, 2001).…”

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

“…The near lethal phenotype of srs2 Δ sgs1 Δ and srs2 Δ rad54 Δ double mutants is restored by deletion of Rad51, suggesting that lethality stems from toxic recombination intermediates (Ira et al, 2003; Klein, 2001). Indeed, Srs2 is well established as a prototypical anti-recombinase due to its ability to remove Rad51 from ssDNA (Antony et al, 2009; Krejci et al, 2003; Marini and Krejci, 2010; Niu and Klein, 2016; Qiu et al, 2013; Sasanuma et al, 2013; Vasianovich et al, 2017; Veaute et al, 2003). Srs2 is orthologous to E. coli UvrD helicase, which can remove the bacterial recombinase RecA from ssDNA (Petrova et al, 2015; Veaute et al, 2005).…”

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

“…Srs2 is an ATP-dependent 3′→5′ ssDNA motor protein that plays a central role in minimizing cross-overs during mitosis (2, [10][11][12][13][14][15][16][17]. Srs2 functions by dismantling Rad51-ssDNA and D-loop intermediates, thereby channeling HR intermediates through the synthesis-dependent strand annealing (SDSA) pathway, which exclusively yields non-cross-over recombination products ( Fig.…”

“…Srs2 functions by dismantling Rad51-ssDNA and D-loop intermediates, thereby channeling HR intermediates through the synthesis-dependent strand annealing (SDSA) pathway, which exclusively yields non-cross-over recombination products ( Fig. 1A) (2, [10][11][12][13][14][15][16]. Consequently, srs2Δ mutants have a mitotic hyperrecombination phenotype characterized by frequent crossovers and gross chromosomal rearrangements (15,16,(18)(19)(20).…”