A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination.
In the yeast Saccharomyces cerevisiae, recombination between direct repeats is synergistically reduced in rad1 rad52 double mutants, suggesting that the two genes define alternate recombination pathways. Using a classical genetic approach, we searched for suppressors of the recombination defect in the double mutant. One mutation that restores wild-type levels of recombination was isolated. Cloning by complementation and subsequent physical and genetic analysis revealed that it maps to RAF1. This locus encodes the large subunit of the single-stranded DNA-binding protein complex, RP-A, which is conserved from S. cerevisiae to humans. The rfa1 mutation on its own causes a 15-fold increase in direct-repeat recombination. However, unlike most other hyperrecombination mutations, the elevated levels in rfa1 mutants occur independently of RAD52 function. Additionally, rfa1 mutant strains grow slowly, are UV sensitive, and exhibit decreased levels of heteroallelic recombination. DNA sequence analysis of rfa1 revealed a missense mutation that alters a conserved residue of the protein (aspartic acid 228 to tyrosine [D228Y]). Biochemical analysis suggests that this defect results in decreased levels of RP-A in mutant strains. Overexpression of the mutant subunit completely suppresses the UV sensitivity and partially suppresses the recombination phenotype. We propose that the defective complex fails to interact properly with components of the repair, replication, and recombination machinery. Further, this may permit the bypass of the recombination defect of rad1 rad52 mutants by activating an alternative single-stranded DNA degradation pathway.
Smith
J
J
Department of Genetics & Development, College of Physicians & Surgeons, Columbia University, New York, New York 10032.
Rothstein
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005838:Genotype; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051721:Rad52 DNA Repair and Recombination Protein; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D051716:Replication Protein A; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D011994:Recombinant Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins