Semidominant Mutations in the Yeast Rad51 Protein and Their Relationships with the Srs2 Helicase

Abstract

Suppressors of the methyl methanesulfonate sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase turned out to contain semidominant mutations in Rad51, a homolog of the bacterial RecA protein. The nature of these mutations was determined by direct sequencing. The 26 mutations characterized were single base substitutions leading to amino acid replacements at 18 different sites. The great majority of these sites (75%) are conserved in the family of RecA-like proteins, and 10 of them affect sites corresponding to amino acids in RecA that are probably directly involved in ATP reactions, binding, and/or hydrolysis. Six mutations are in domains thought to be involved in interaction between monomers; they may also affect ATP reactions. By themselves, all the alleles confer a rad51 null phenotype. When heterozygous, however, they are, to varying degrees, negative semidominant for radiation sensitivity; presumably the mutant proteins are coassembled with wild-type Rad51 and poison the resulting nucleofilaments or recombination complexes. This negative effect is partially suppressed by an SRS2 deletion, which supports the hypothesis that Srs2 reverses recombination structures that contain either mutated proteins or numerous DNA lesions.The RAD51 gene of Saccharomyces cerevisiae is involved in recombination and recombinational repair (for a review, see reference 19). It encodes a protein with homologies to the bacterial RecA proteins (1,3,36). These homologies are localized in the regions that form the hydrophobic core of the RecA protein, containing the ATPase and DNA binding domains, as deduced from the three-dimensional structure of the molecule (38-40).Rad51 forms with double-stranded (ds) DNA (27) and single-stranded (ss) DNA (42) filaments similar in structure to those observed with RecA. In the presence of the Rpa DNAbinding proteins, it catalyzes the homologous DNA strand exchange reaction (41,42). Three other S. cerevisiae genes, RAD55 (20), RAD57 (16), and DMC1 (7), also code for proteins with homologies to the ATPase domain of RecA. RAD51, RAD55, and RAD57 belong to the RAD52 epistasis group of genes involved in the repair of ionizing radiation (10) and are part of the same homologous recombination pathway (29). These proteins may well be part of a multiprotein complex: Rad51 interacts with Rad52 in vitro (36) and in vivo (24); it also interacts with Rad55, which in turn interacts with Rad57 (11,15). DMC1 is expressed only during meiosis and is involved in meiotic recombination (7). The Dmc1 and Rad51 proteins appear to be associated in recombination complexes (6). Homologs of Rad51 have also been identified in two other yeasts, Kluyveromyces lactis (9) and Schizosaccharomyces pombe (14, 35), in Neurospora crassa (8), and in higher eucaryotes, including Xenopus laevis (21), chicken (5), mouse (26,35), and human cells (35). In all cases, the regions of greatest homology are confined to the ATPase and DNA binding domains. The human protein forms nucleofilaments with both ss and ds DNAs (4).In ...

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…From genetic studies we know that SRS2 can be placed at a stage where it counteracts recombination (Chanet et al, 1996;Rong et al, 1991) and SGS1 downstream, at its resolution (Fabre et al, 2002;Ira et al, 2003).…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…Srs2 possesses a 3′ to 5′ DNA helicase activity [66,67] that is crucial for recombination [67] and suppression of DDT defects [68,69]. Genetic data indicate that Srs2 negatively regulates recombination [70,71] possibly by reversal of intermediate recombination structures [72][73][74][75]. Indeed, the DNA strand exchange mediated by Rad51 is inhibited by Srs2 through disruption of the Rad51-ssDNA filaments [76,77], and it turns out that sumoylated PCNA has increased affinity for Srs2 [78,79] and represses the Rad52-dependent recombination pathway [80].…”

Role of ATP Hydrolysis in the Antirecombinase Function of Saccharomyces cerevisiae Srs2 Protein

“…Rad51 associated with the magnetic bead-bound duplex is eluted by SDS followed by SDS-PAGE analysis. B, pre-assembled Rad51-ssDNA nucleoprotein filaments (lanes 2-8) were incubated with Srs2 (WT, 60 and 90 nM in lanes 3 and 4), srs2 K41A (K/A, 90 and 180 nM in lanes 5 and 6), or srs2 K41R (K/R, 90 and 180 nM in lanes 7 and 8), and the reaction mixtures mixed with streptavidin magnetic beads containing biotinylated dsDNA (lanes [3][4][5][6][7][8]. In lane 1, free Rad51 was mixed with streptavidin magnetic beads that contained biotinylated dsDNA, and in lane 2, free Rad51 was mixed with streptavidin magnetic beads that did not contain any dsDNA.…”

“…Accordingly, srs2 mutants are sensitive to DNA damaging agents and show a hyperrecombination phenotype. Genetic deletion of the RAD51 or RAD52, key members of the RAD52 epistasis group functioning in homologous recombination, alleviates the DNA damage sensitivity and hyperrecombination phenotypes of srs2 mutants (8), implicating untimely recombination events as the progenitor of these srs2 phenotypes. Srs2 mutations are lethal when combined with mutations in a variety of genes needed for DNA repair and other chromosomal processes, e.g.…”

“…One such helicase is encoded by the Saccharomyces cerevisiae SRS2 gene, altered forms of which were first described as either suppressors of the DNA damage sensitivity of rad6 and rad18 mutants (6) or as hyperrecombination mutants (7). Detailed genetic analyses have shown that a major function of SRS2 is to attenuate homologous recombination activity to allow for the channeling of certain DNA lesions into the RAD6/ RAD18-mediated postreplication repair pathway (8,9). Accordingly, srs2 mutants are sensitive to DNA damaging agents and show a hyperrecombination phenotype.…”