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Molecular and Cellular Biology, January 2000, p. 224-232, Vol. 20, No. 1
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Naz Erdenizdagger and Rodney Rothstein*

Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032-2704

Received 6 August 1999/Returned for modification 13 September 1999/Accepted 23 September 1999

In Saccharomyces cerevisiae, RAD1 and RAD52 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 a RAD52-independent recombination pathway. To identify gene(s) acting in this pathway, temperature-sensitive (ts) mutations were screened for those that decrease recombination levels in a rad1 rad52 rfa1-D228Y strain. Three mutants were isolated. Each segregates as a single recessive gene. Two are allelic to RSP5, 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. 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 wild-type strains. We propose that Rsp5 is involved in the degradation of Rfa1 linking ubiquitination with the replication-recombination machinery.

* Corresponding author. Mailing address: Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 West 168th St., New York, NY 10032-2704. Phone: (212) 305-1733. Fax: (212) 923-2090. E-mail: rothstein{at}cuccfa.ccc.columbia.edu.

dagger Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

Molecular and Cellular Biology, January 2000, p. 224-232, Vol. 20, No. 1
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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