A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.
To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endonuclease. Besides isolating a number of new alleles in known rad genes, we identified a novel allele of the RFA1 gene, rfa1-44, which encodes the large subunit of the heterotrimeric yeast single-stranded DNA-binding protein RPA. Characterization of rfa1-44 revealed that it is, like members of the RAD52 epistasis group, sensitive to X rays, high doses of UV, and HO-induced DSBs. In addition, rfa1-44 shows a reduced ability to undergo sporulation and HO-induced gene conversion. The mutation was mapped to a single-base substitution resulting in an aspartate at amino acid residue 77 instead of glycine. Moreover, all radiation sensitivities and repair defects of rfa1-44 are suppressed by RAD52 in a dose-dependent manner, and one RAD52 mutant allele, rad52-34, displays nonallelic noncomplementation when crossed with rfa1-44. Presented is a model accounting for this genetic interaction in which Rfa1, in a complex with Rad52, serves to assemble other proteins of the recombination-repair machinery at the site of DSBs and other kinds of DNA damage. We believe that our findings and those of J. Smith and R. Rothstein (Mol. Cell. Biol. 15:1632-1641, 1995) are the first in vivo demonstrations of the involvement of a eukaryotic single-stranded binding protein in recombination and repair processes.
Firmenich
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Department of Biochemistry, Beckman Center for Molecular & Genetic Medicine, Stanford University School of Medicine, California 94305.
Elias-Arnanz
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Berg
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D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005785:Gene Conversion; D005800:Genes, Fungal; D005838:Genotype; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D051721:Rad52 DNA Repair and Recombination Protein; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D011995:Recombination, Genetic; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D013172:Spores, Fungal / Q000502:physiology; D013489:Suppression, Genetic; D014466:Ultraviolet Rays; D014965:X-Rays
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D011994:Recombinant Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific