A new type of radiation-sensitive mutant of S. cerevisiae is described. The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase. Homozygous diploids are as sensitive as G1 haploids. The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased. The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes. The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa. All the consensus domains of known DNA helicases are present. Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E. coli were found. The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase.
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 ...
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 endjoining 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.Mutations in the Saccharomyces cerevisiae SRS2 gene were isolated in different genetic screens, namely suppression of the trimethoprim sensitivity of rad6 and rad18 mutants (29), suppression of the DNA damage sensitivity in rad18 cells (2), and hyperrecombination (3). The Srs2 protein shares homologies with the bacterial UvrD, Rep (2), and PcrA helicases. As predicted by the sequence, Srs2 was shown in vitro to have 3Ј-5Ј DNA helicase activity (50).Genetic studies showed that a number of deleterious phenotypes associated with srs2 null alleles are suppressed by deletions of the RAD51, RAD52, RAD55, and RAD57 genes, which are all involved in the formation of Rad51 nucleofilaments on single-stranded DNA (ssDNA) (1, 9, 26). These suppressed phenotypes include UV sensitivity and synthetic lethality with rad54 (17, 47, 52) and sgs1 (14) mutations, which both (most likely) affect later recombination steps (4,13,38). Links between Srs2 and homologous recombination were also revealed by the fact that srs2 null alleles suppress the ␥-ray and methyl methanesulfonate (MMS) sensitivities conferred by several leaky alleles of RAD52 and RAD51 but have no suppressor effects on the corresponding deletions (9, 25, 42, 52). These phenotypes led to the proposal that Srs2 could destabilize recombination intermediates poisoned to some extent by the leaky recombination proteins (9). In the absence of Srs2, the leakiness of the proteins would allow efficient recombinational repair to occur. In support of this interpretation, Srs2 was recently shown to disrupt in vitro Rad51 nucleofilaments on ssDNA (27,66). This activity might well account for the srs2 phenotypes cited above, which can be explaine...
The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase delta, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain. To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13. Mutations in genes encoding the two other subunits of DNA polymerase delta (HYS2, POL32) were identified. Mutations in two recombination genes (RAD50, RAD51) were also identified, confirming that homologous recombination is necessary for pol3-13 mutant strain survival. Other mutations were identified in genes involved in repair and genome stability (MET18/ MMS19), in the control of origin-firing and/or transcription (ABF1, SRB7), in the S/G2 checkpoint (RAD53), in the Ras-cAMP signal transduction pathway (MKS1), in nuclear pore metabolism (SEH1), in protein degradation (DOC1) and in folding (YDJ1). Finally, mutations in three genes of unknown function were isolated (NBP35, DRE2, TAH18). Synthetic lethality between pol3-13 and each of the three mutants pol32, mms19 and doc1 could be suppressed by a rad18 deletion, suggesting an important role of ubiquitination in DNA replication control. We propose that the pol3-13 mutant generates replicative problems that need both homologous recombination and an intact checkpoint machinery to be overcome.
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