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 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.
Erdeniz
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N
Department of Genetics, College of Physicians and Surgeons, Columbia University, New York, New York 10032-2704, USA.
Rothstein
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R
D000483:Alleles; D000595:Amino Acid Sequence; D045643:DNA Repair Enzymes; D004271:DNA, Fungal / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C469287:RSP5 protein, S cerevisiae; D045643:DNA Repair Enzymes