Rsp5, a ubiquitin-protein ligase, is involved in degradation of the single-stranded-DNA binding protein Rfa1 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, RAD1 and RAD52 are required for alternate path ways of mitotic recombination, Double-mutant strains exhibit a synergistic interaction that decreases direct repeat recombination rates dramatically. A mutation in RFA1, the largest subunit of a single-stranded DNA-binding pr otein complex (RP-A), suppresses the recombination deficiency of rad1 rad52 strains (J. Smith and R. Rothstein, Mol. Cell. Biol, 15:1632-1641, 1995), Previously, we hypothesized that this mutation, rfa1-D228Y, causes an incre ase in recombinogenic lesions as well as the activation of a RAD52-independ ent recombination pathway. To identify gene(s) acting in this pathway, temp erature-sensitive (ts) mutations were screened for those that decrease reco mbination levels in a rad1 rad52 rfa1-D228Y strain. Three mutants were isol ated. Each segregates as a single recessive gene, Two are allelic to RSP5, which encodes an essential ubiquitin-protein ligase. One allele, rsp5-25, c ontains two mutations within its open reading frame. The first mutation doe s not alter the amino acid sequence of Rsp5, but it decreases the amount of full-length protein in vivo. The second mutation results in the substituti on of a tryptophan with a leucine residue in the ubiquitination domain, In rsp5-25 mutants, the UV sensitivity of rfa1-D228Y is suppressed to the same level as in strains overexpressing Rfa1-D228Y, Measurement of the relative rate of protein turnover demonstrated that the half-life of Rfa1-D228Y in rsp5-25 mutants was extended to 65 min compared to a 35-min half-life in wi ld-type strains. We propose that Rsp5 is involved in the degradation of Rfa 1 linking ubiquitination with the replication-recombination machinery.