Suchergebnisse

DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation. ; Research was funded by grants from the Spanish Ministry of Science and Innovation (BFU2010-16372 and Consolider Ingenio 2010 CSD2007-0015), the Junta de Andalucía (BIO-102 and P09/CVI4567), and the European Union (FEDER) to AA and from the National Institutes of Health (GM50237 and GM67055) to RR. ; Peer reviewed

While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs. ; The Spanish Ministry of Science and Innovation [BFU2006-05260, BFU2010-16372 and Consolider Ingenio 2010 CSD2007-015 to A.A., SAF2010-21017 to F.C.L. and SAF2010-14877 to P.H.]; Junta de Andalucía [BIO102 and CVI4567] and the European Union (FEDER) (to A.A.); Cancer Research UK [C6/A11224]; the European Community's Seventh Framework Programme (FP7/2007-2013) [HEALTH-F2-2010-259893 (DDResponse)] and ERC Advanced Researcher (to S.P.J.). Funding for core infrastructure in the SPJ lab was provided by Cancer Research UK [C6946/A14492] and the Wellcome Trust [092096]. S.P.J. receives his salary from the University of Cambridge, supplemented by Cancer Research UK. S.M.G. and A.L.S. were recipient of predoctoral training fellowships from the Spanish Research Council (CSIC) and the Spanish Ministry of Science and Innovation (FPI fellowship), respectively. Funding for open access charge: Consolider Ingenio 2010 grant CSD2007-015. ; Peer reviewed