Abstract
The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.
This work was supported by grants from the Centre National de la Recherche Scientifique (UMR2027 CNRS/I.C and UMR217 CEA/CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Association pour la Recherche sur le Cancer (ARC), the Institut Curie and the Commissariat à l'Energie Atomique (CEA). Christine Soustelle was supported by a postdoctoral fellowship from CEA.
Thanks are due to Serge Urbach for his expert advice about immunodetection, to Patrick Hughes for his comments and corrections on the manuscript, to Serge Gangloff for the pSG plasmids, to Mark Hochstasser for the Smt3 antibody, and to Natalie Declerck for help in using Rasmol. We also thank Gérard Faye and Giuseppe Baldacci for helpful discussions about this work.