Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. © 2013 European Molecular Biology Organization

Optimal reaction conditions for the 3′–5′ exonuclease activity of the Ape2

<p><b>Copyright information:</b></p><p>Taken from "Human Ape2 protein has a 3′–5′ exonuclease activity that acts preferentially on mismatched base pairs"</p><p>Nucleic Acids Research 2006;34(9):2508-2515.</p><p>Published online 10 May 2006</p><p>PMCID:PMC1459411.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The 3′–5′ exonuclease activity of GST–Ape2 was assayed under various reaction conditions using a partial DNA duplex (S3) (10 nM) in which the 5′-labeled oligonucleotide contained a 3′-recessed terminus. () Metal ion dependence. The first lane contains reaction mixture without any metal ions in the reaction buffer. Reactions were carried in the presence of 8 mM MgCl (lanes 2–4) or 0.5 mM MnCl (lanes 5–7) and increasing concentrations of Ape2 as indicated. () Graphical representation of results in (A); () NaCl concentration dependence. The first lane contains reaction without any Ape2 protein. () Graphical representation of results in (C); () pH dependence. () Graphical representation of results in (E)

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

International audienceCompletion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability

Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover

International audienceIn vegetative cells, most recombination intermediates are metabolized without an association with a crossover (CO). The avoidance of COs allows for repair and prevents genomic rearrangements, potentially deleterious if the sequences involved are at ectopic locations. We have designed a system that permits to screen spontaneous intragenic recombination events in Saccharomyces cerevisiae and to investigate the CO outcome in different genetic contexts. We have analyzed the CO outcome in the absence of the Srs2 and Sgs1 helicases, DNA damage checkpoint proteins as well as in a mutant proliferating cell nuclear antigen (PCNA) and found that they all contribute to genome stability. Remarkably high effects on COs are mediated by srs2Δ, mrc1Δ and a pol30‐RR mutation in PCNA. Our results support the view that Mrc1 plays a specific role in DNA replication, promoting the Srs2 recruitment to PCNA independently of checkpoint signaling. Srs2 would prevent formation of double Holliday junctions (dHJs) and thus CO formation. Sgs1 also negatively regulates CO formation but through a different process that resolves dHJs to yield non‐CO products