Copyright 2001 by the Genetics Society of America Dissection of the Functions of the Saccharomyces cerevisiae RAD6 Postreplicative Repair Group in Mutagenesis and UV Sensitivity Petr Cˇ ejka,*,† Vladimı´r Vondrejs* and Zuzana Storchova´*,† *Department of Genetics and Microbiology, Faculty of Natural Sciences, Charles University, 128 44 Prague, Czech Republic and †Institute of Medical Radiobiology, University of Zurich, 8008 Zurich, Switzerland Manuscript received May 9, 2001 Accepted for publication August 17, 2001 ABSTRACT The RAD6 postreplicative repair group participates in various processes of DNA metabolism. To elucidate the contribution of RAD6 to starvation-associated mutagenesis, which occurs in nongrowing cells cultivated under selective conditions, we analyzed the phenotype of strains expressing various alleles of the RAD6 gene and single and multiple mutants of the RAD6, RAD5, RAD18, REV3, and MMS2 genes from the RAD6 repair group. Our results show that the RAD6 repair pathway is also active in starving cells and its contribution to starvation-associated mutagenesis is similar to that of spontaneous mutagenesis. Epistatic analysis based on both spontaneous and starvation-associated mutagenesis and UV sensitivity showed that the RAD6 repair group consists of distinct repair pathways of different relative importance requiring, besides the presence of Rad6, also either Rad18 or Rad5 or both. We postulate the existence of four pathways: (1) nonmutagenic Rad5/Rad6/Rad18, (2) mutagenic Rad5/Rad6 /Rev3, (3) mutagenic Rad6/ Rad18/Rev3, and (4) Rad6/Rad18/Rad30. Furthermore, we show that the high mutation rate observed in rad6 mutants is caused by a mutator different from Rev3. From our data and data previously published, we suggest a role for Rad6 in DNA repair and mutagenesis and propose a model for the RAD6 postreplicative repair group. UTATIONS play a fundamental role in evolution One of the DNA repair pathways suggested to be in- M and contribute to aging, carcinogenesis, and ge- volved in SAM of the unicellular eukaryotic organism netic diseases. Spontaneous mutations occur during Saccharomyces cerevisiae is the RAD6 postreplicative repair DNA replication by incorrect nucleotide incorporation, pathway (Storchova´ et al. 1998). This pathway is in- followed by skipping the proofreading activity of replica- volved in the repair of UV-induced DNA lesions and tive polymerases and the activity of the mismatch repair other bulky lesions that block DNA replication and in pathway; arise as DNA repair errors; or are introduced mutagenesis. The RAD6 epistatic group can be dissected by some mutagenic system. Mutations resulting after into various repair subpathways, but they are poorly mutagenic treatment are called induced mutations and understood (Liefshitz et al. 1998; Xiao et al. 2000). appear to result from mutagenic repair (Friedberg et The pivotal gene of this group is RAD6. Its product al. 1995). In the past decade, it was shown that mutations functions in various cellular processes including DNA also occur during prolonged nonlethal starvation in repair and mutagenesis, gene silencing, protein degra- nongrowing cells in both bacteria and unicellular eu- dation, sporulation, and histone H2B ubiquitination. karyotes. These mutations are called adaptive or star- Null rad6 mutants exhibit a pleiotropic phenotype—they vation associated. Several mechanisms have been sug- possess a defect in all of the above-listed functions—that gested for adaptive mutations in bacteria (e.g., Foster contributes to their extreme sensitivity to various DNA- 2000). They are suggested to be mainly a result of incor- damaging agents, enhanced spontaneous and impaired rect DNA repair of endogenous lesions arising in starv- induced mutagenesis, lower growth rate, decreased via- ing cells (e.g., Bridges 1996); however, the existence bility under stress conditions, and so on (Lawrence 1994). of such lesions has not yet been substantiated. The abil- The protein Rad6 consists of 172 amino acid residues, ity of cells to generate mutations in even a quiescent from which the last 23 form an almost entirely acidic state appears to be a general phenomenon, at least C-terminal tail (Morrison et al. 1988). It is a ubiquitin- among unicellular organisms. However, we do not un- conjugating enzyme (E2) catalyzing the ubiquitination derstand the exact mechanism of starvation-associated of proteins in cooperation with other proteins. Rad6 mutagenesis (SAM) and we do not know its contribution ubiquitinates either by forming a Lys-48 polyubiquitin to survival and evolution of microorganisms. chain, which serves as a signal for proteosomal degrada- tion of the ubiquitylated proteins (Dohmen et al. 1991; Watkins et al. 1993), or by monoubiquitination, which Corresponding author: Zuzana Storchova´, Institute of Medical Radiobi- ology, University of Zurich, August Forel Str. 7, 8008 Zurich, Switzer- is not a signal for degradation and is known, for exam- land. E-mail: [email protected] ple, for histones (Robzyk et al. 2000). The protein was Genetics 159: 953–963 (November 2001) 954 P. Cˇ ejka, V. Vondrejs and Z. Storchova´ shown to interact tightly with either Ubr1 for ubiquitin- nonessential DNA polymerase has the capability to by- mediated N-end rule protein degradation (Watkins et pass thymine dimers and other replication-blocking le- al. 1993) or Rad18 for the DNA repair functions (Bailly sions at the cost of an increased mutation frequency et al. 1997a,b). (translesion synthesis; Nelson et al. 1996; Baynton et According to mutational analysis, the functions of the al. 1999). The role of the Rad6/Rad18 heterodimer in Rad6 protein can be attributed to its distinct domains. this process is not very clear, but it was suggested that The first nine amino acids are required for interaction it allows the recruitment of pol␨ to the stalled replication with Ubr1 and thus for N-end rule-mediated protein fork (Bailly et al. 1997b). degradation (Watkins et al. 1993). They are probably On the basis of recent epistatic studies, RAD30 belongs also involved in error-free repair (Broomfield et al. to neither of the two above-mentioned subpathways and 1998). The amino acids 142–149 and, less importantly, probably represents a third subpathway (Xiao et al. residues 10–22 are responsible for the interaction with 2000). It encodes translesion polymerase ␩ that exhibits Rad18 and thus for the DNA repair functions of Rad6 low fidelity and tolerance to DNA damage (Washing- (Bailly et al. 1997a,b). Cysteine at position 88 is re- ton et al. 1999). The significance of this pathway in quired for binding of a ubiquitin molecule (Sung et al. DNA repair remains unclear. 1990). The acidic C-terminal tail is involved in ubiquiti- We have shown previously that the rad6-1 mutation nation of histone H2B, sporulation, meiotic functions, significantly enhanced SAM (Storchova´ et al. 1998). and, less importantly, in nonspecific interaction with To clarify the role of Rad6 in SAM in more detail, we Ubr1 (Sung et al. 1988; Robzyk et al. 2000; Ulrich and constructed a rad6 null mutant and complemented the Jentsch 2000). mutation by various rad6 alleles carried on plasmids. As mentioned above, Rad6 interacts with Rad18, the The analysis showed that, in particular, Rad6 DNA re- product of another member of the RAD6 group. This pair function is responsible for maintaining the low level interaction is necessary for Rad6 DNA repair functions, of SAM in the wild-type strain. We therefore analyzed because Rad18, unlike Rad6, shows an affinity to ssDNA the effect of deletion of various RAD6 repair group and appears to target Rad6 toward damaged DNA genes on spontaneous and starvation-associated muta- (Bailly et al. 1994). The Rad6/Rad18 complex has genesis and sensitivity to UV light to elucidate the role been assumed to be required for all recognized subpath- of the error-free and error-prone subpathways in SAM. ways within the RAD6 group (see below), although rad18 Our experimental system allowed us to compare the mutants do not show a DNA repair deficiency as strong relative importance of various RAD6 group genes in as rad6 mutants. SAM and spontaneous mutagenesis. The analysis of rev3, Epistasis studies have identified three subpathways rad5, rad18, and mms2 single and multiple mutants al- within this group up to now. An error-free pathway lowed us to propose a model for RAD6-mediated sub- acting by a yet-unknown mechanism involves Rad5, pathways acting in repair and mutagenic processes. Mms2, Ubc13, Pol30, and pol␦ (Xiao et al. 1999, 2000). One of the members, Rad5, has DNA helicase and zinc- MATERIALS AND METHODS binding domains, shows affinity to ssDNA, and tran- siently interacts with the ubiquitin-conjugating protein General methods: For nonselective growth, YPD medium complex Mms2/Ubc13 (Johnson et al. 1994; Ulrich consisting of 2% glucose, 1% bactopeptone, and 0.5% yeast and Jentsch 2000). The products of MMS2 and UBC13 extract was used (all Difco, Detroit). For selection of clones with replacement of the gene of interest by kanMX4 cassette, have both been shown to be members of the RAD6 YPD with G418 (400 ␮g/ml; GIBCO BRL, Paisley, Scotland) epistasis group and involved in error-free repair and was used. Synthetic dropout (SD) medium (0.75% yeast nitro- form a tight heterodimer possessing the ability to conju- gen base without amino acids, 2% glucose, and dropout solu- gate a polyubiquitin chain in vitro via an unusual Lys- tion; Difco) was used for selective growth when the essential 63 (Hofmann and Pickart 1999). In vitro studies also supplement for selection was omitted. All the media were solidified in 2% agar (Difco). Yeast genetics methods were showed interaction of Rad5 and Rad18. It was suggested used essentially as described (Ausubel et al. 1994). All yeast that the two similar ubiquitin-conjugating enzymes Rad6 strains were propagated under aerobic conditions at 30Њ. and Mms2/Ubc13 form complexes with either Rad18 Yeast strains: The yeast strains used in this study for analysis or Rad5, respectively.