Copyright 0 1990 by the Genetics Society o f America

The SRS2 Suppressor of rad6 Mutations of Saccharomyces cerevisiae Acts by Channeling DNA Lesions Into theRAD52 DNA Repair Pathway Robert H. Schiestl,**’Satya Prakash* and Louise Prakasht *Department of Biology, University of Rochester, Rochester, New York 14627 and tDepartment o f Biophysics, University of Rochester School of Medicine, Rochester, New York 14642 Manuscript received October 24, 1989 Accepted for publication December 22, 1989 ABSTRACT rad6 mutants of Saccharomyces cerevisiaeare defective in the repair of damaged DNA, DNAdamage inducedmutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors ofthe UV sensitivity of rad6 deletion (rad6A) mutations and show that they also suppress the y-ray sensitivity but notthe U V mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of y-ray sensitivity. The six suppressor mutations we isolated are all alleles of thesame locus and arealso allelic to a previously described suppressor of therad6-1 nonsense mutation, SRS2. We show that suppression of rad6A is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6A SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into theRAD52 DNA repair pathway are discussed.

T

HE RAD6 gene of Saccharomyces cerevisiae is required for a variety of cellular functions. rad6 mutants are highly sensitive to DNA damaging agents including UV, ionizing radiation,and alkylating agents (HAYNES and KUNZ 1981; GAME1983). Inrad6 mutants, DNA strand discontinuities, formed during DNA replication in the newly synthesized DNA strand across fromnon-coding lesions, arenotrepaired (PRAKASH1981). rad6 mutants are defective in mutagenesis induced by a variety of DNA damaging agents (PRAKASH 1974; LAWRENCE and CHRISTENSEN 1976; LAWRENCE1982)andare also defective in meiotic recombination and sporulation (GAMEet al. 1980; MONTELONE, PRAKASH and PRAKASH 1981). In addition, rad6 mutants grow poorly and have reduced plating efficiency. RAD6 encodes a proteinof 172 aminoacids of 19.7 kD, which contains a highly acidic carboxyl terminus in which 20 of the 23 amino acids are acidic (REYNOLDS, WEBERand PRAKASH 1985). Deletion of the polyacidic domain of RAD6 causes a defect in sporulation but not in the DNA repair and mutagenesis functions (MORRISON,MILLER and PRAKASH1988). RAD6 is a ubiquitin conjugating (E2) enzyme which has been shown toconjugate ubiquitin to histones H2A and H2Bin vitro (JENTSCH,MCGRATHand VARSHAVSKY 1987; SUNG, PRAKASH and PRAKASH1988). I Present address: Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599. T h e publication costs o f this article were partly defrayed by the payment of page charges. This article nust therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. $1734 solely to indicate this fact.

Genetics 1 2 4 817-831 (April, 1990)

T o identify DNA repair genes that can substitute for the absence of R A D 6 function, we have isolated suppressors of the UV sensitivity of a r a d 6 A strain in which the entire genomic RAD6 gene has been deleted. Such suppressor mutations could arise in genes which, when overexpressed, replace the missing RAD6 activity. For example, increased amounts of another ubiquitin conjugating enzyme, such as that encoded by the CDC34 gene (GOEBLet al. 1988), might substitute for the missing RAD6 function. One could also obtain bypass suppressor mutations which act by providing access to an alternate pathway that can substitute for the blocked pathway. Suppressor analysis has been immensely useful in the identification of new recombination genes in Escherichia coli. CLARKand his colleagues isolated the sbcA and sbcB suppressors of the recombination deficiency in recBC mutants (BARBOUR et al. 1970; KUSHNER et al. 197 1). A new ATP independent exonuclease activity (exoVIII) is overproduced in sbcA mutants (KUSHNER, NAGAISHI and CLARK1974), and loss of exonuclease I activity occurs in sbcB mutants (KUSHNERet al. 197 1). Isolation of mutations that cause recombination deficiency in the recBC sbcB strain revealed the existence of the RecF pathway of recombination that can substitute for theRecBC recombination pathway in the sbcA or sbcB mutant background (HORIIand CLARK1973; SMITH1988). The RecE, RecF, RecJ, RecN, RecO,RecQ and R u v genes participate in the RecF recombination pathway (HORIIand CLARK1973; SMITH1988). All the suppressors of the r a d 6 A mutation we isolated are alleles of one locus and they are allelic to the

R. H. Schiestl, S. Prakash and L. Prakash

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