Dissection of the Functions of the Saccharomyces Cerevisiae RAD6 Postreplicative Repair Group in Mutagenesis and UV Sensitivity

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 degradation 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 transiently 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. These complexes are held to- of reversion of the suppressible amber ade2-101 allele were gether by interaction between Rad5 and Rad18 and derivatives of YPH499/500 strains (Sikorski and Hieter 1989) and are listed in Table 1. The replacement of RAD6, bind DNA (Ulrich and Jentsch 2000). Furthermore, REV3, RAD5, and MMS2 was performed using a kanMX4 re- POL30, encoding proliferating cell nuclear antigen placement cassette obtained by PCR with specifically designed (PCNA), also belongs to this group. It probably acts with primers using pUG6 plasmid as a template (Guldener et al. pol␨ to perform DNA synthesis after repair of lesions 1996). Sequences of primers used are available on request. (Torres-Ramos et al. 1996). The disruption of RAD18 with the functional LEU2 gene was performed via homologous recombination of the BamHI-HpaI The key role in a second mutagenic subpathway is fragment from YCp50-11 plasmid (Fabre et al. 1989), kindly played by polymerase ␨, encoded by REV3 and REV7 provided by F. Fabre. Yeast transformation was performed as (catalytic and regulatory subunits, respectively). This previously described (Gietz et al. 1995). Transformants were RAD6 Epistatic Group in Mutagenesis 955

TABLE 1 Saccharomyces cerevisiae strains

Strain Genotype Source YPH499 MATa ura3-52 lys2-801 ade2-101 leu2-⌬1 his3-⌬200 trp1-⌬63 Sikorski and Hieter (1989) YPH500 MAT␣ ura3-52 lys2-801 ade2-101 leu2-⌬1 his3-⌬200 trp1-⌬63 Sikorski and Hieter (1989) YCP1 YPH499 rad6⌬::KANMX4 This study YCP2 YPH499 rad18::LEU2 This study YCP3 YPH499 rad6⌬::KANMX4 rad18::LEU2 This study YCP4 YPH499 rev3⌬::KANMX4 This study YCP6 YPH500 rev3⌬::KANMX4 This study YCP7 YPH499 rev3⌬::KANMX4 rad18::LEU2 This study YCP8 YPH500 rad5⌬::KANMX4 This study YCP9 YPH500 mms2⌬::KANMX4 This study YCP11 YPH499 rad5⌬::KANMX4 rad6⌬::KANMX4 This study YCP12 YPH499 rad5⌬::KANMX4 rad18::LEU2 This study YCP13 YPH500 rad5⌬::KANMX4 rad6⌬::KANMX4 rad18::LEU2 This study YCP14 YPH499 rev3⌬::KANMX4 rad6⌬::KANMX4 This study YCP15 YPH499 rev3⌬::KANMX4 rad5⌬::KANMX4 This study

streaked on appropriate omission medium and single colonies onto SD plates supplemented with adenine to determine the were picked for further analysis. All mutants were verified number of colony-forming units (cfu). Revertants to Adeϩ both genomically using PCR and/or Southern blotting and prototrophy were counted daily from the 3rd to the 12th day phenotypically using a UV-sensitivity test. Multiple mutants of selective starvation. The growth-dependent mutation rate were prepared by crossing single mutant strains followed by was calculated according to Lea and Coulson (1949). tetrad dissection. We have always constructed and tested two Cell survival analysis: Yeast cultures cultivated nonselectively to three independent isolates to reduce the chance of sponta- up to a density of 2 ϫ 108 cells/ml were harvested, washed, ϫ 105 cells were spotted 5ف neously occurring suppressors appearing in the genetic back- and diluted. Drops containing ground. All details about strain construction are available on onto SD plates lacking adenine. The agar cubes with drops request. were cut out in regular intervals, washed, diluted if necessary, Plasmid manipulation: Plasmids carrying various alleles of and plated onto SD plates supplemented with adenine to score the RAD6 gene (Bailly et al. 1997a) were kindly provided by total survivors. In the case of the strains containing plasmids, S. Prakash. The control plasmids were prepared by excision the appropriate supplement was omitted to reveal only the of the RAD6 open reading frame from the plasmids. For the number of living cells with plasmid. Each result is an average list of given plasmids see Table 2. of at least three independent experiments. Reversion in ade2-1: Spontaneous mutation rates and accu- UV killing analysis: The late exponential phase cultures mulation of starvation-associated Adeϩ revertants were deter- were harvested, washed, and diluted. Proper dilutions were mined by conducting a modified fluctuation test. Three-day- plated on YPD plates and irradiated (UV Stratalinker 1800). old colonies (10–24 independent isolates) were transferred The number of colonies was counted after 4 days of cultivation to liquid media and grown nonselectively up to a density of in a dark chamber. Each result is an average of three to five 2 ϫ 108 cells/ml, harvested, and washed with sterile water. independent experiments. Each culture was plated on SD plates lacking adenine (usually Evaluation of starvation-associated mutagenesis: The data 5 ϫ 107 cells per plate) and after appropriate dilution also describing mutagenesis and viability in stationary phase are

TABLE 2 Plasmids used in this study

Plasmid Description Source pR67 CEN, URA3, RAD6 Sung et al. 1990 ␮ pR6.33 2 , URA3, rad6⌬165-172 Bailly et al. (1997b) ␮ pR6.32 2 , URA3, rad6⌬154-172 Bailly et al. (1997b) ␮ pR6.31 2 , URA3, rad6⌬150-172 Bailly et al. (1997b) pTB248 2␮, URA3, rad6-1 Bailly et al. (1997b) ␮ pR6.61 2 , TRP1, ADC1::rad6⌬1-9 Watkins et al. (1993) ␮ pR648 2 , TRP1, ADC1::rad6Ala88 Sung et al. (1990) pLH CEN, URA3 L. Hlavata´ pPCU 2␮, URA3 This study pPCT 2␮, TRP1 This study YCp50-11 CEN, URA3, RAD18 Fabre et al. (1989) prad18⌬ rad18::LEU2 Fabre et al. (1989) 956 P. Cˇ ejka, V. Vondrejs and Z. Storchova´ presented in three different graphics in Figures 1, 3, and 6. of a visible colony takes, on average, 3 days for the wild- In Figures 1A, 3A, and 6A, the accumulation curve shows the type strain YPH499 (parameter T ϭ 3; see materials cumulative average of the total number of revertant colonies that appeared during selective starvation. The data were nor- and methods), whereas the period required for the days (D ϭ 3.5ف of the colonies was %95ف malized to 108 cfu on the day of plating. Figures 1B, 3B, formation of and 6B show the percentage of colony-forming units plotted 3.5). The various mutant strains derived from YPH499 against the time of starvation. And Figures 1C, 3C, and 6C grew similarly (T ϭ 3; D ϭ 3.5) except for the rad6⌬ show both accumulation and viability data combined. It shows strain and all strains carrying the rad6 mutation in com- the hypothetical number of starvation-associated revertant colonies per 108 living cells during the whole period of starvation. bination with any other mutation, as well as strains car- Because the numbers are noncumulative, it shows the increase rying the rad6Ala88 allele (T ϭ 3.5; D ϭ 4). Thus, we of revertant colonies in defined intervals. The basic formula considered all the colonies arising before day D to be used for calculating the transformed number of revertant colo- a result of spontaneous events occurring during nonse- ϭ nies after t days of starvation is nt nt(100/cfutϪT), where nt lective growth before plating for selection, and the is the observed number of revertant colonies after t days of counted numbers were used for calculation of the spon- starvation and cfutϪT is the percentage of colony-forming units after tϪT days of starvation. T (days) accounts for the average taneous mutation rates. All colonies arising later are a delay between the mutation event and the observation of a result of starvation-associated mutagenesis. visible colony. We have also established a factor D (days), To describe starvation-associated mutagenesis we fol- ف which accounts for the interval when 95% of the cells form lowed the accumulation of revertants (Figures 1A, 3A, visible colonies under corresponding experimental conditions (presence of nongrowing background cells). Note the differ- and 6A) and cell viability (Figures 1B, 3B, and 6B) of colonies) and D during prolonged starvation. In Figures 1C, 3C, and %50ف ence between T (formation of Figures 1C, 3C, and 6C show only revertants that 6C, both data are combined, and a daily increment of .(%95ف) appeared after D days of starvation—the starvation-associated starvation-associated revertant colonies is plotted in a revertants. The curve is noncumulative. Further details about noncumulative manner. Thus, Figures 1C, 3C, and 6C data analysis are available on request. show numbers not influenced by the loss of viability during starvation. This approach allows us to compare RESULTS AND DISCUSSION the strains even if they differ in their growth, viability, and in the number of preexisting revertants. For a de- The experimental system for analysis of spontaneous tailed description of the evaluation of our experimental and SA mutagenesis: Yeast auxotrophic strains can form data, see materials and methods. two types of revertant colonies when plated on plates The effect of RAD6 alleles on SAM suggests an lacking an essential supplement. The first type of colony involvement of Rad6 repair functions: We have shown ف appears early, on average 3–4 days after plating. The previously that a mutation in the RAD6 gene increases corresponding mutations occurred very likely by replica- the level of starvation-associated mutagenesis (Stor- tion errors during the nonselective growth before plat- chova´ et al. 1998). Because Rad6 acts in a number of ing. The second type of revertant colony appears later various cellular processes, we used the defined alleles, and accumulates up to 20 days as a consequence of whose products possess a well-known lack of function, mutation events occurring during the starvation on se- to elucidate in more detail the role of Rad6 in SAM. lective plates (e.g., Hall 1992; Steele and Jinks-Rob- As in the experimental approach, we complemented ertson 1992; Storchova´ et al. 1998). the chromosomal RAD6 deletion with a plasmid carrying The evaluation of late revertant accumulation is com- one of the various alleles of RAD6 (Table 2). In strains plicated by two facts: overexpressing mutant proteins we followed spontane- 1. The cells are losing their colony-forming ability dur- ous mutagenesis and SAM. Because the analyzed alleles were carried on plasmids, we also performed all the ing starvation, and individual mutant strains differ ⌬ in their sensitivity to starvation. This effect strongly necessary controls with rad6 and wild-type strains trans- influences the number of arising revertants. formed with plasmids lacking the coding sequence for 2. It is difficult to estimate the proportion of preexisting any of the Rad6 mutant proteins. There were no signifi- cant differences in mutation rates among transformed and starvation-associated (SA) revertants, because al- ⌬ ⌬ though the colonies of SA revertants arise later, there strains, not between rad6 strain and rad6 transformed is a certain interval at the beginning of starvation with plasmids without the coding sequence for various when both types of revertant colonies arise. RAD6 alleles or between wild-type strain and wild type transformed with the control plasmids (Table 3). We We performed a reconstruction experiment, which was observed slight differences in cell viability during starva- suggested to address this problem (Steele and Jinks- tion that accounted for slight differences in the accu- Robertson 1992). In this experiment the formation of mulation of starvation-associated revertants, which were visible colonies was followed on complete SD plates with caused by differential loss of plasmids under stress (data .(ϫ 107 nongrowing nonrevertible not shown 5ف a background of cells, which slows down the colony appearance. We have The expression of Rad6 protein with deletions of the ascertained that under these conditions the formation acidic C terminus, varying in length up to the amino RAD6 Epistatic Group in Mutagenesis 957

TABLE 3 Spontaneous mutation rates of overexpressed alleles carried on plasmids

Strain Transformed with plasmid Resulting genotype Rate (ϫ10Ϫ8) Relative to wild type YPH499 None wt 2.0 (0.80, 2.4) 1 YCP1 None rad6 8.9 (5.7, 14) 4.5 YCP1 pR67 RAD6 2.6 (1.4, 4.7) 1.3

YCP1 pR6.33 rad6⌬165-172 2.5 (1.9, 3.6) 1.3 YCP1 pR6.32 rad6⌬154-172 2.4 (1.1, 3.9) 1.2 YCP1 pR6.31 rad6⌬150-172 2.6 (1.4, 3.4) 1.3 YCP1 pTB248 rad6-1 13 (9.4, 17) 6.5

YCP1 pR6.61 rad6⌬1-9 9.9 (8.7, 13) 5.0 YCP1 pR648 rad6Ala88 5.9 (3.1, 8.8) 3.0 YCP1 pPCT rad6 14 (7.9, 18) 7.0 YCP1 pPCU rad6 11 (8.5, 12) 5.3 YCP1 pLH rad6 13 (9.9, 20) 6.5 YPH499 pPCT wt 2 (1.3, 2.8) 1 YPH499 pPCU wt 1.7 (1.1, 2.9) 0.9 YPH499 pLH wt 1.2 (0.9, 2.1) 0.6 The mutation rate was calculated according to Lea and Coulson (1949). Low and high quartiles for each sample are given in parentheses. acid 150, complemented the rad6⌬ phenotype to the changed by the presence of the rad6-1 allele encoding wild-type level in both starvation-associated (Figure 1, a protein shortened from the C terminus to residue A and C) and spontaneous mutagenesis (Table 3). The 142. The truncated protein is very likely able to ubiquiti- viability of these strains during starvation is also compa- nate but it cannot interact tightly with Rad18, because rable with the wild type (Figure 1B). The C-terminal this interaction occurs via the missing eight amino acids part of Rad6 was shown to be involved in direct ubiquiti- 142–149 (Bailly et al. 1997a,b). Because Rad18, unlike nation of H2B by Rad6 (Robzyk et al. 2000), and mu- Rad6, possesses an ssDNA-binding activity and probably tated strains also had a reduced sporulation efﬁciency targets Rad6p toward damaged DNA, the truncated (Morrison et al. 1988). The N-end rule protein degra- Rad6 protein is deﬁcient in a majority of its DNA repair dation is decreased, because the C terminus of Rad6 is functions (Bailly et al. 1997a,b). The rad6-1 strain is also involved in interaction with Ubr1 (Watkins et al. only mildly sensitive to a starvation, and combination 1993). Thus, all these functions are dispensable for main- of the accumulation and viability curves reveals a high taining a low level of spontaneous and SA mutagenesis. increase in the number of SA revertants (Figure 1). The mutation frequency in the rad6⌬ strain was not The rad6⌬1-9 allele encodes the Rad6 protein lacking

Figure 1.—Accumulation of revertants (A), viability (B), and daily noncumulative increment of SAM (C) during prolonged starvation of strains overexpressing various RAD6 alleles. Wild type (ϫ); rad6 (ᮀ); ᭹ ᭝ RAD6 ( ); rad6⌬150-172 ( ); rad6-1 ᭡ ᭛ ᭺ ( ); rad6⌬1-9 ( ); and rad6Ala88 ( ). For A, 95% confidence interval of the total number of revertants (day 12) represents the following: wild type (Ϯ11.6); rad6 (Ϯ20.2); RAD6 (Ϯ9.8); Ϯ Ϯ rad6⌬150-172 ( 5.0); rad6-1 ( 21.0); Ϯ Ϯ rad6⌬1-9 ( 22.0); rad6Ala88 ( 24.0). 958 P. Cˇ ejka, V. Vondrejs and Z. Storchova´ the first nine amino acids, which are necessary for the Rad6/Ubr1 interaction (Watkins et al. 1993). The strain overproducing this allele behaves similarly to the rad6-1 strain and shows both increased spontaneous (Ta- ble 3) and SA mutagenesis together with moderate starvation sensitivity (Figure 1). Mutant rad6⌬1-9 was shown to have impaired Ubr1-dependent ubiquitination function, which labels certain proteins for proteosomal degradation (Sung et al. 1990). Because the proteosomal mutants do not have increased UV sensitivity or mutage- nicity (Dor et al. 1996), we can conclude that the observed increase of SA spontaneous mutagenesis in this mutant is not due to impaired proteosomal degradation. Mutant rad6⌬1-9 was also shown to be sensitive to UV, and UV-induced mutagenesis in this mutant was as high as in the wild type (Watkins et al. 1993). This suggests that the first nine amino acids are essential for the error- free repair but dispensable for the error-prone repair. The increased level of spontaneous and SA mutagenesis reflects a preferential repair of DNA lesions by the error- prone repair pathway, because the error-free pathways in the rad6⌬1-9 strain are inactive. → The strain expressing rad6-Ala88 (mutation Cys88 Figure 2.—UV sensitivity of strains overexpressing various Ala88) produces the Rad6 protein that is completely ϫ ᮀ ᭹ RAD6 alleles. Wild type ( ); rad6 ( ); RAD6 ( ); rad6⌬150-172 ᭝ ᭡ ᭛ ᭺ lacking ubiquitin-conjugating activities (Sung et al. 1990). ( ); rad6-1 ( ); rad6⌬1-9 ( ); and rad6Ala88 ( ). This strain and the rad6⌬ mutant show a very similar phenotype, consistent with the generally accepted hypothesis that ubiquitin-conjugating activities are re- alleles: The strains carrying various plasmid-borne RAD6 quired for all the RAD6 functions (Broomfield et al. alleles were also analyzed for their UV sensitivity (Figure 1998). Both rad6⌬ and rad6-Ala88 strains are extremely 2). The alleles coding for protein with the acidic tail sensitive to starvation, which in combination with a deleted ensure the same UV resistance as wild-type Rad6 rather strong mutator phenotype, leads to fast revertant protein. Mutants rad6⌬1-9 and rad6-1 are markedly less accumulation up to the sixth or seventh day of starva- sensitive to UV irradiation than rad6-Ala88 and rad6⌬ tion, followed by considerable slowing down of revertant mutants. We hypothesize that this difference is caused accumulation (Figure 1). Very likely the dramatic de- by remaining DNA repair activity of proteins coded by crease of viability during starvation prevents further for- rad6⌬1-9 and rad6-1, because it was shown that both of mation of revertant colonies. The rad6⌬ and rad6-Ala88 them can at least partially ensure the repair functions mutants have the same phenotype in spontaneous and (Dor et al. 1996). The absence of ubiquitination in both starvation-associated mutagenesis that rad6-1 and rad6 rad6-Ala88 and rad6⌬ mutants results in a very strong ⌬1-9 strains have (Figure 1C). However, the different phenotype independently of the presence or absence sensitivity to starvation is notable: while rad6⌬ and rad6- of the protein. These two mutants are similarly sensitive Ala88 are extremely sensitive, rad6-1 and rad6⌬1-9 show to starvation. Thus, the phenotypes observed with high only moderate sensitivity. The complex phenotype of sensitivity to stress are probably caused by loss of other the rad6⌬ and rad6-Ala88 strains is very likely a conse- cellular RAD6 functions, whereas in rad6⌬1-9 and rad6-1 quence of the simultaneous damage of many different mutants the observed phenotypes are very likely only the biological processes. The increased mutagenesis in consequence of a deficiency in RAD6 repair functions. rad6⌬1-9, rad6-1, rad6-Ala88, and rad6⌬ is probably due Epistatic relationships between RAD6, RAD5, and to inefficient RAD6-dependent error-free repair. Thus, RAD18: To analyze the role of various genes from the the RAD6-dependent error-free functions are required RAD6 epistatic group, we created a set of strains with for maintaining a low level of both starvation-associated chromosomal deletions of genes of interest (Table 1) mutations and spontaneous mutations. and analyzed spontaneous and starvation-associated mu- The observed phenotypes could be a consequence of tagenesis in each strain. After analyzing single mutants, an overexpression of the mutant rad6 alleles. However, we observed a similar spontaneous mutation rate in rad5 because Rad6 is rather abundant in yeast cells during mutant as in rad6 mutant (Table 4). The spontaneous the entire cell cycle, this effect appears to be highly mutation rate of rad18 strain was slightly, but not sig- unlikely (Dor et al. 1996). nificantly, lower. UV sensitivity of strains overexpressing various RAD6 The rad5 rad18 double mutant has a very low mutation RAD6 Epistatic Group in Mutagenesis 959

TABLE 4 show the same sensitivity. Also, the double-mutant rad5 Spontaneous mutation rates of analyzed mutants rad18 is as sensitive as the rad6 strain as well as the rad5 rad6 rad18 triple mutant. Thus, the RAD6 is epistatic Relevant Relative to and the effects of RAD18 and RAD5 are synergistic. For Strain genotype Rate (ϫ10Ϫ8) wild type repair, RAD6 and either RAD18 or RAD5 are required. The repair is mutagenic in the absence of RAD6 and/ YPH499 wt 2.0 (0.80, 2.4) 1 YCP1 rad6 8.9 (5.7, 14) 4.5 or in the absence of either RAD18 or RAD5 or nonmuta- YCP2 rad18 7.8 (6.2, 11) 3.9 genic in the absence of both RAD5 and RAD18. YCP8 rad5 9.9 (8.7, 24) 4.5 The rad5 and rad18, but not rad6, mutator phenotype YCP4 rev3 1.6 (1.0, 2.3) 0.8 is completely dependent on REV3: The important muta- YCP9 mms2 13 (9.7, 28) 6.5 tor in the RAD6 repair group appears to be the DNA -of sponta %60ف YCP3 rad6 rad18 7.8 (6.2, 11) 3.9 polymerase ␨, which is responsible for YCP11 rad6 rad5 9.5 (6.9, 13) 4.8 neous mutations (Roche et al. 1994). We analyzed the YCP12 rad5 rad18 3.3 (2.3, 4.8) 1.7 YCP13 rad5 rad6 rad18 6.6 (4.0, 9.3) 3.3 strain with a chromosomal deletion of REV3, which YCP7 rev3 rad18 1.3 (0.8, 1.7) 0.7 codes for the catalytic subunit of this mutagenic polymer- YCP14 rev3 rad6 7.9 (5.4, 11) 4.0 ase. Rev3 is expected to act downstream of Rad6 in a YCP15 rev3 rad5 1.6 (0.9, 2.1) 0.8 Rad6-dependent manner (Lawrence and Christen- sen 1976). The rev3 strain shows a low level of spontaneous mutagenesis (Table 4) as previously described rate, comparable with wild type (Table 4). However, the (Roche et al. 1995). The mutagenic effect of the rad18 triple rad6 rad18 rad5 mutant shows a similar spontane- deletion is completely dependent on Rev3 functions ous mutation rate as any double mutant in combination because the mutation rate of the rad18 rev3 double mu- with the rad6 mutation. We assume that the mutagenic tant is as low as that of the rev3 single mutant (Table 4). repair mediated by Rad6 requires the presence of either Similarly, the rad5 rev3 mutant shows the same mutation Rad18 or Rad5. In the rad5 rad18 double mutant and rate as rev3. This means that Rev3 can act either in in the presence of Rad6, the repair is channeled into a subpathway mediated by Rad18 or in a subpathway a different error-free repair pathway, which results in mediated by Rad5. However, the mutator phenotype of low mutagenesis. This assumption can be applied also rad6 is only slightly decreased by the rev3 deletion and, to SA mutagenesis because the epistasis follows exactly according to the statistical analysis, it is statistically indis- the same pattern (Figure 3). tinguishable from rad6, rad5 rad6,orrad18 rad6 mutant The UV-sensitivity analysis supports these data (Figure strains. It is interesting that rad6 shows any mutator 4). The rad5 and rad18 single mutants are moderately phenotype, because the pol␨ activity has been consid- sensitive; the rad18 strain is more sensitive than a rad5 ered to be completely dependent on Rad6 function mutant. The sensitivity of the rad6 strain is very high, (Lawrence and Christensen 1976). Our results clearly and the rad6 rad5 and rad6 rad18 double-mutant strains show that the cause of a higher mutation rate in rad6

Figure 3.—Accumulation of revertants (A), viability (B), and daily noncumulative increment of SAM (C) of rad5, rad6, and rad18 single-, double-, and triple-mutant strains. Wild type (ϫ); rad6 (ᮀ); rad18 (᭝); rad5 (᭺); mms2 (᭜); rad6 rad18 (᭛); rad5 rad6 (᭿); rad5 rad18 (᭹); and rad5 rad6 rad18 (᭡). For A, 95% con- ﬁdence interval of the total number of revertants (day 12) represents the following: wild type (Ϯ11.6); rad6 (Ϯ20.2); rad18 (Ϯ21.7); rad5 (Ϯ32.8); mms2 (Ϯ45.5); rad6 rad18 (Ϯ24.8); rad5 rad6 (Ϯ18.8); rad5 rad18 (Ϯ15.0); and rad5 rad6 rad18 (Ϯ37.7). 960 P. Cˇ ejka, V. Vondrejs and Z. Storchova´

Figure 5.—The epistasis of rev3, rad5, rad6, and rad18 single Figure 4.—UV sensitivity of rad5, rad6, and rad18 single-, ϫ double-, and triple-mutant strains. Wild type (ϫ); rad6 (ᮀ); and double mutants in UV sensitivity. Wild type ( ); rad6 ᭝ ᭺ ᭜ ᭛ (ᮀ); rad18 (᭝); rad5 (᭺); rev3 (ϩ); rev3 rad18 (—); rev3 rad6 rad18 ( ); rad5 ( ); mms2 ( ); rad6 rad18 ( ); rad5 rad6 ᭜ (᭿); rad5 rad18 (᭹); and rad5 rad6 rad18 (᭡). ( ); and rev3 rad5 (*).

␨ dent on the RAD6 repair pathway and the second is mutants is unrelated to polymerase . Another situation independent of RAD6. is observed in UV-induced mutagenesis, where the rad6 MMS2 belongs to the error-free pathway: The highest and rev3 strains show the same antimutator phenotype mutation rate was observed in the mms2 mutant (Table (Kunz et al. 2000). 4). This confirms the engagement of MMS2 in the error Which pathways are responsible for the high muta- free branch of RAD6 repair pathway (Broomfield et al. genesis rate in rad6 and rad6 rev3 mutants? We propose 1998). This strain was considered as a control for the the existence of another mutagenic repair pathway dif- situation when only error-prone pathways of RAD6 re- ferent from Rev3-mediated repair. This pathway is repair pathways are functional. Also the SAM is very high sponsible for nearly all mutations in rad6 null mutants in the mms2 strain, confirming the hypothesis that an and all multiple mutants in combination with rad6 and error-free RAD6 repair pathway is responsible for main- does not occur if at least partially functional Rad6 pro- taining the low level of SAM (Figure 3). tein is present. Recently, it was shown that POL32 might A single mms2 mutant shows very low sensitivity to UV be responsible for the mutator phenotype of rad6 null and at low doses of UV it is almost indistinguishable mutant, because the double mutant rad6 pol32 shows from the wild-type strain (Figure 4). Thus, this protein nearly no mutations in the canavanine resistance for- has a more important function in avoiding mutagenic ward assay (Huang et al. 2000). repair than in the repair of UV-induced lesions. The analysis of UV sensitivity revealed the additive The mutants show a similar phenotype in both starva- sensitivity in double mutants rad5 rev3 and rad18 rev3 tion-associated and spontaneous mutagenesis: The ef- (Figure 5), suggesting that RAD5 and REV3, and RAD18 fect in starvation-associated mutagenesis of the mutants and REV3, respectively, form two separate and indepen- analyzed follows basically the same principles as in spon- dent DNA repair pathways. It also confirmed the parity taneous mutagenesis. Our findings might suggest that of RAD5- and RAD18-mediated subpathways. We also spontaneous and starvation-associated mutagenesis can- observed that the relative sensitivity decreases with not be shown to be different processes. However, it was higher levels of irradiation in the rad18 rev3 mutant already shown that the type of reversion and mutation strain (Figure 5). This would suggest the existence of spectrum is different in spontaneous and SA mutagene- an unknown DNA repair activity induced by a high level sis (Storchova´ et al. 1998; Heidenreich and Win- of DNA damage. tersberger 2001). We suppose that the RAD6 postrepli- In contrast to previously published results, our de- cative repair pathway is a nonspecific pathway ensuring tailed analysis revealed higher UV sensitivity of rad6 rev3 tolerance to any blocking lesion in DNA and thus help- mutants than a single rad6 mutant, suggesting that Rev3 ing to maintain chromosomal stability. Such a function could work in two different modes. The first is depen- can be important in any stage of the yeast life cycle, RAD6 Epistatic Group in Mutagenesis 961

Figure 6.—Accumulation of revertants (A), viability (B), and daily noncumulative increment of SAM (C) of rad5, rad6, and rad18 mutants and its dependency on rev3 mutation. Wild type (ϫ); rad6 (ᮀ); rad18 (᭝); rad5 (᭺); rev3 (ϩ); rev3 rad18 (—); rev3 rad6 (᭜); and rev3 rad5 (*). For A, 95% conﬁdence interval of the total number of revertants (day 12) represents the following: wild type (Ϯ11.6); rad6 (Ϯ20.2); rad18 (Ϯ21.7); rad5 (Ϯ32.8); rev3 (Ϯ4.2); rev3 rad18 (Ϯ6.2); rev3 rad6 (Ϯ12); and rev3 rad5 (Ϯ6.1).

which results in similar epistasis observed for both spon- efficiently removed from DNA. Our data on the rev3 taneous and starvation-associated mutagenesis. The RAD6 mutant support the latter hypothesis. repair pathway is believed to be activated as the replica- Overall, it appears that the genetic relationships in tion machinery stalls when it is unable to bypass a lesion spontaneous and SA mutagenesis are similar. An impor- to further synthesize DNA (for review, see Kunz et al. tant exception is the rev3 rad18 double mutant, which 2000). We assume that this happens not only during shows an unusual course in the accumulation of starva- conventional DNA replication in growing cells, but also tion-associated revertants. At the beginning, the muta- during repair synthesis in starving and nongrowing cells. genesis is very low, as in wild-type and rev3 mutants. The The strains can be classified into three groups ac- numbers of revertants increase later with the period of cording to the accumulation of starvation-associated re- starvation and finally reach levels as high as in rad5 vertants: (1) the mms2, rad6, rad5, and rad18 single mu- or rad18 single mutants (Figure 3C). This suggests the tants, the rad6 rad5, rad6 rad18, and rad6 rev3 double possible induction of another DNA repair pathway act- mutants, and the rad6 rad5 rad18 triple mutant show ing in a mutagenic manner. The mutant also reveals the highest level of SAM, similar to spontaneous muta- very unusual survival after UV irradiation: it is very sensi- genesis; (2) relatively low levels of revertants, again simi- tive to low-dose UV irradiation, but relatively less sensi- lar to spontaneous mutagenesis, are seen in the rad5 tive to the higher doses. Induced activation of an alterna- rad18 double mutant; and (3) the lowest numbers of SA tive pathway as a result of severe DNA damage caused revertants were observed in wild-type, rev3, and rev3rad5 by UV irradiation or long-term starvation might explain strains. Although the number of SA revertants in the this phenomenon. This pathway is very likely not medi- rev3 strain is significantly lower in comparison to the wild ated by another error-prone polymerase coded by RAD30 type, the mutant strain is less viable during starvation, so because its activity requires the Rad6/Rad18 hetero- the resulting SA mutagenesis of the rev3 strain is only dimer (Washington et al. 1999). partially lower than that of the wild type (Figure 6). RAD6 repair pathway is active in starving cells: Al- Polymerase ␨ is synthesized at the same level during the though stationary phase cells have 10-fold less RAD6 entire cell cycle (Singhal et al. 1992), unlike major mRNA than exponential phase cells (Madura et al. replicative DNA polymerases (Morrison et al. 1988; 1990), our results suggest that the RAD6 repair pathway Johnson et al. 1994), and thus its effect could be higher is active in nondividing cells. The Rad6 protein is rather in the stationary phase. Hence, this polymerase was sug- abundant in the exponential phase and its concentra- gested to play a key role in starvation-associated mutation is not limiting the DNA repair (unlike the concen- genesis as a result of mutagenic synthesis either on un- tration of Rad18; Dor et al. 1996). Therefore, even a damaged templates (Holbeck and Strathern 1997) substantial decrease of Rad6 concentration can sustain or during translesion synthesis (Baynton et al. 1998). activity of the RAD6 repair pathway. Furthermore, we However, our results surprisingly suggest that the role confirmed the presence of RAD6 mRNA in S. cerevisiae of error-prone polymerase ␨ in the mutagenesis of non- even after 5 days of starvation in liquid culture without a growing cells is not higher than in spontaneous muta- single supplement (Z. Storchova´, unpublished data). genesis. The starvation-associated mutations can arise Model for RAD6 postreplicative repair group: We in the wild-type strain either because more mutations showed that the Rad6 protein acts in two different ways are formed during starvation or because they are less during DNA repair and mutagenesis: (1) it functions 962 P. Cˇ ejka, V. Vondrejs and Z. Storchova´

Figure 7.—Model of interactions within the RAD6 repair group. in both error-free and error-prone DNA repair pathways either Rev3 or Rad30. It was shown that Rad30 can in cooperation with Rad18 and/or Rad5 and (2) it inter- be involved in both error-free and error-prone repair, feres with other DNA repair pathways. For example, the depending on lesion type (Yuan et al. 2000), and its phenotype of the rad5 rad18 mutant is different in the activity definitely requires Rad18 and Rad6 (McDonald presence or absence of RAD6. Both rad5 and rad18 sin- et al. 1997). This is not the case for Rev3, because the gle mutants exhibit a very high mutation rate, almost mutator phenotype of both rad5 and rad18 mutants is exclusively dependent on REV3. Deletion of both genes completely dependent on Rev3. Thus, pol␨ is functional brings about a dramatic decrease in mutagenesis and a in the Rad18/Rad6-mediated subpathway as well as in synergistic effect on UV sensitivity, which suggests par- the Rad5/Rad6 subpathway. The Rad6/Rad5/Rev3 sub- tial redundancy of Rad5- and Rad18-mediated pathways pathway is responsible for the majority of mutations in and competition for the same substrate, which might be rad18 strains, whereas the Rad6/Rad18/Rev3 subpath- processed by REV3 if any of Rad5 or Rad18 are present. way is responsible for the mutations in rad5 strains. However, further deletion of rad6 increases the muta- The model presented above is based almost exclu- tion rate of the rad5 rad18 double mutant again. The sively on genetic analysis, which presupposes linear un- presence of the Rad6 protein might therefore block branched processes. To the best of our knowledge, the other repair pathways. scarce biochemical data available are in agreement with On the basis of our genetic data and data published our model. Our study underlines the need for more by other groups, we suggest the following working detailed biochemical studies, which will be necessary to model for the RAD6 postreplicative group. We hypothe- perform to understand the exact role of the RAD6 repair size that the RAD6 repair group consists of three differ- pathway in both growing and starving cells. ent subpathways (Figure 7). The first complex is respon- We thank S. Prakash, F. Fabre, and L. Hlavata´ for plasmids used in sible for major error-free repair and consists of Rad18/ this study. Z.S. is grateful to P. Scha¨r and M. Ra¨schle for comprehensive Rad6/Rad5/Mms2/Ubc13 proteins allowing the error- suggestions about the manuscript and Ann E. Randolph for English free bypass of lesions in DNA. All these proteins were grammar corrections. This work was supported partially by the Grant shown to interact at least transiently (Ulrich and Agency of Charles University 1999/293 B BIO to Z.S. and partially by the Institute of Medical Radiobiology with kind support of J. Jiricny. Jentsch 2000). MMS2, UBC13, pol␦, and PCNA were clearly shown to belong only to the error-free pathway (Torres-Ramos et al. 1996; Broomfield et al. 1998; LITERATURE CITED Hofmann and Pickart 1999). Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seid- The second subpathway requires Rad18/Rad6 dimers man, J. A. Smith and K. Struhl, 1994 Current Protocols in Molecu- and ensures that translesion synthesis is performed by lar Biology. John Wiley & Sons, New York. RAD6 Epistatic Group in Mutagenesis 963

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