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Firmenich, AA, Elias-Arnanz, M and Berg, P (1995) A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52. Mol. Cell. Biol. 15:1620-31

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.

PubMed PMC230386

Alleles; Base Sequence; Cloning, Molecular; Crosses, Genetic; DNA Repair; DNA-Binding Proteins/biosynthesis; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Deoxyribonucleases, Type II Site-Specific/metabolism; Fungal Proteins/genetics; Fungal Proteins/metabolism; Gene Conversion; Genes, Fungal; Genotype; Macromolecular Substances; Molecular Sequence Data; Mutagenesis; Oligodeoxyribonucleotides; Plasmids; Rad52 DNA Repair and Recombination Protein; Recombinant Proteins/biosynthesis; Recombinant Proteins/metabolism; Recombination, Genetic; Replication Protein A; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae/physiology; Saccharomyces cerevisiae Proteins; Spores, Fungal/physiology; Suppression, Genetic; Ultraviolet Rays; X-Rays