Rsp5, a Ubiquitin-Protein Ligase, Is Involved in Degradation of the Single-Stranded-DNA Binding Protein Rfa1 in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, RAD1 andRAD52 are required for alternate pathways 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 protein 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 increase in recombinogenic lesions as well as the activation of aRAD52-independent recombination pathway. To identify gene(s) acting in this pathway, temperature-sensitive (ts) mutations were screened for those that decrease recombination levels in arad1 rad52 rfa1-D228Y strain. Three mutants were isolated. Each segregates as a single recessive gene. Two are allelic toRSP5, which encodes an essential ubiquitin-protein ligase. One allele, rsp5-25, contains two mutations within its open reading frame. The first mutation does 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 substitution of a tryptophan with a leucine residue in the ubiquitination domain. Inrsp5-25 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 wild-type strains. We propose that Rsp5 is involved in the degradation of Rfa1 linking ubiquitination with the replication-recombination machinery.

ACKNOWLEDGMENTS

We thank Uffe Mortensen, Steve Sturley, Xiaolan Zhao, and especially Marcel Wehrli for comments on the manuscript and helpful discussions. We also thank Jon Huibregtse, Carl Mann, Bruce Stillman, and Steve Brill for the kind gifts of strains, plasmids, and antibodies. Finally, we thank Adam Bailis, Serge Gangloff, John McDonald, Julie Smith, and Hui Zou for encouragement and discussion throughout the course of this work.

This work was supported by National Institutes of Health grant GM50237 (R.R.).