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.
Mesh Terms:
Alleles, Base Sequence, Cloning, Molecular, Crosses, Genetic, DNA Repair, DNA-Binding Proteins, Deoxyribonucleases, Type II Site-Specific, Fungal Proteins, Gene Conversion, Genes, Fungal, Genotype, Macromolecular Substances, Molecular Sequence Data, Mutagenesis, Oligodeoxyribonucleotides, Plasmids, Rad52 DNA Repair and Recombination Protein, Recombinant Proteins, Recombination, Genetic, Replication Protein A, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spores, Fungal, Suppression, Genetic, Ultraviolet Rays, X-Rays
Mol. Cell. Biol.
Date: Mar. 01, 1995
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