Delineating the Requirements for Spontaneous DNA Damage Resistance Pathways in Genome Maintenance and Viability in Saccharomyces cerevisiae

Corresponding author: Sue Jinks-Robertson, Emory University, 1510 Clifton Rd., Atlanta, GA 30322., jinks{at}biology.emory.edu (E-mail)

Communicating editor: A. NICOLAS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cellular metabolic processes constantly generate reactive species that damage DNA. To counteract this relentless assault, cells have developed multiple pathways to resist damage. The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal. Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways. In this study, we examine the consequences of simultaneously compromising three or more of these pathways. Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal. In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth. In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions.

SPONTANEOUS DNA lesions occur frequently and at such a level that cells have developed multiple pathways to resist DNA damage by either removing the damage or bypassing the lesion until it can be repaired at a later time. Early yeast genetic studies examining epistatic relationships among mutant rad alleles conferring radiation sensitivity defined the nucleotide excision repair (NER; the RAD3 epistasis group) pathway, the recombination (REC; the RAD52 epistasis group) pathway, and the postreplication repair (PRR; the RAD6 epistasis group) pathway, which is composed of several mutagenic translesion synthesis (TLS) and error-free subpathways (for reviews of these pathways, see PRAKASH and PRAKASH 2000 ; SUNG et al. 2000 ; BROOMFIELD et al. 2001 ). The importance of base excision repair (BER) in the processing of DNA damage was not examined in the early yeast genetic studies as the classic rad mutants do not impact this pathway. More recent studies, however, have shown that mutants deficient in BER and either NER, the error-prone component [DNA polymerase (Pol )] of PRR, or REC are viable and exhibit the phenotypes expected for overlapping DNA damage resistance specificities (SWANSON et al. 1999 ; TORRES-RAMOS et al. 2000 ; GELLON et al. 2001 ).

Although BER, like NER, is a DNA damage excision pathway, BER is very different from NER in the specificity of the lesions recognized as well as in the enzymatic process of damage removal (for a review of BER, see MEMISOGLU and SAMSON 2000A ). In BER, specific N-glycosylases recognize various base lesions, many of which are generated during normal cellular metabolism (reviewed in DIANOV et al. 2001 ). The abasic site resulting from N-glycosylase-mediated excision of the damaged base can be processed by either an AP endonuclease or an AP lyase, resulting in cleavage of the sugar-phosphate DNA backbone. Subsequent reactions involving a DNA polymerase can fill in a single nucleotide (short-patch BER) or six to seven nucleotides (long-patch BER). In contrast to BER, NER uses an enzyme complex to recognize a broad range of DNA lesions (reviewed in PRAKASH and PRAKASH 2000 ). After the initial recognition, BER and NER are further distinguished by the extent of DNA removed, with NER making dual incisions to initiate the removal of a 25- to 30-nucleotide patch, which is then filled in by DNA polymerase. As alternatives to damage removal, the REC and PRR pathways function as DNA damage bypass pathways, which allow DNA damage to be tolerated until it can be removed at a later time. In addition to lesion bypass, the REC pathway also functions in the repair of single- and double-strand breaks (reviewed in CROMIE et al. 2001 ).

The inability to remove mutational intermediates and the response of cells to DNA damage often affects haploid and diploid yeast strains differently. Simultaneous loss of the proofreading activities of DNA polymerases and is lethal in haploids, presumably resulting in an error "catastrophe," but is tolerated in diploids (MORRISON and SUGINO 1994 ). Likewise, mismatch repair deficiency in addition to DNA polymerase proofreading deficiency is lethal only in haploids (MORRISON et al. 1993 ). In terms of induced DNA damage, exposure of haploids to ultraviolet radiation in late S/G2 of the cell cycle results in greater survival than exposure in the G1/S phase (CHANET et al. 1973 ; SIEDE and FRIEDBERG 1990 ), and diploids show a greatly increased resistance to -radiation compared to haploids (BEAM et al. 1954 ; reviewed in SAEKI et al. 1980 ; GAME 1993 ). In both cases it has been argued that enhanced survival is due to the presence of a second genomic complement, which can be used for recombinational repair. Differential ploidy sensitivity has also been reported with additional DNA-damaging agents (ARMAN and DUTOVA 1975 ; SAKOVICH and EFREMOV 1978 ; MONDON and SHAHIN 1992 ) as well as with overexpression of the recombination protein Rad54p (CLEVER et al. 1999 ).

In our earlier studies, some of the haploid mutants deficient in two of the four resistance pathways exhibited poor growth, suggesting that viability might be partially compromised (SWANSON et al. 1999 ). In the current study, we examine the extent to which individual DNA damage resistance pathways (BER, NER, PRR, and REC) can support growth in haploid and homozygous diploid yeast strains. The results indicate that of the four DNA damage resistance pathways, only BER can support haploid growth in the absence of the other three pathways. In contrast, BER, NER, or REC alone can support growth in a diploid, but the PRR pathway alone cannot. These results affirm the overlapping roles of the yeast DNA damage resistance pathways in responding to spontaneous DNA damage and reveal the central role of the BER pathway in this process.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and growth conditions:
Yeast strains were grown nonselectively in YEPD medium (1% yeast extract, 2% Bacto peptone, 2% dextrose, 2% agar for plates) and selectively on synthetic complete (SC) medium (SHERMAN 1991 ) containing 2% dextrose and lacking the appropriate amino acid. Lys⁺ revertants were identified on SC medium lacking lysine (SC-LYS); Can^r mutants were identified on SC medium lacking arginine and supplemented with 60 mg/liter canavanine, and sulfometuron methyl resistant (SM^R) mutants were identified on synthetic minimal medium supplemented with uracil, histidine, and leucine and containing 3 mg/liter sulfometuron methyl (SM; DuPont Agricultural Products; FALCO and DUMAS 1985 ). SC medium containing 1 g/liter 5-fluoroorotic acid (5-FOA; BOEKE et al. 1987 ) was used to select Ura^- segregants. Presence of the bacterial kan gene was detected on YEPD medium containing 200 mg/liter Geneticin (Sigma, St. Louis), while the bacterial hph gene was detected on YEPD medium containing 6 ml/liter hygromycin B (Calbiochem, La Jolla, CA). Diploids were selected on synthetic minimal medium supplemented with uracil, histidine, and leucine as required. To induce sporulation, diploid strains were grown in presporulation medium (1% yeast extract, 2% Bacto peptone, 10% dextrose), washed twice with sterile water, and transferred to sporulation medium (0.22% yeast extract, 0.05% glucose, 2% potassium acetate), supplemented with uracil as needed. Mitotic cultures were grown at 30° and meiotic cultures were grown at room temperature.

Strain constructions:
Yeast transformations were carried out according to GIETZ et al. 1995 with modifications as noted. All strains used in this study are listed in Table 1. Wild-type alleles were replaced with disruption alleles using the following plasmids: NcoI/NdeI-digested pLF298 (ntg1::LEU2; BARTON and KABACK 1994 ); XhoI/SacI-digested pGEM-ntg2::hisG-URA3-hisG (YOU et al. 1998 ); EcoRI/BamHI-digested pSCP19A (apn11::HIS3; RAMOTAR et al. 1991 ); EcoRI/SalI-digested pBRHSURA3 (rad52::URA3; KAYTOR and LIVINGSTON 1994 ); or SalI/EcoRI-digested pR1.6 (rad1::hisG-URA3-hisG; SAPARBAEV et al. 1996 ). When the hisG-URA3-hisG cassette was used for disruption, Ura^- segregants were isolated on 5-FOA. Putative disruption of RAD1 was assayed by UV sensitivity (28 J/m² at 254 nM); disruption of RAD52 was assayed by sensitivity to methyl methanesulfonate (MMS; Kodak, 0.008–0.016% in YEPD plates). UV-irradiated or MMS-containing plates were wrapped in aluminum foil and scored for growth after 2 days.

A PCR-generated rev3::kan disruption fragment was used to delete REV3. Primers 5'-ATGTCGAGGGAGTCGAACGACACAATACAGAGCGATACGGTTAGATCATCCTCTAAATCAcagctgaagcttcgtacg-3' (forward) and 5'-TTACCAATCATTTAGAGATATTAATGCTTCTTCCCTTTGAACAGATTGATTATCTCTCAAaggccactagtgatctg-3' (reverse) were used to amplify an 1-kb disruption fragment using pFA6-kanMX2 (WACH et al. 1994 ) as template. The first 60 bases of each primer are complementary to REV3, and the 3' ends are complementary to the kanamycin resistance cassette (lowercase letters). Following transformation, cells were grown for 3 hr in 2 ml of YEPD before selective plating on YEPD plates containing Geneticin. After 2 days the colonies were replica plated onto fresh Geneticin-containing medium. Likewise, a PCR-generated rad18::kan^R disruption fragment was used to delete RAD18 using primers 5'-ATGGACCACCAAATAACCACTGCAAGCGACTTCACGACTACTTCAATACCGAGCCTGTACcagctgaagcttcgtacg-3' (forward) and 5'-TTAATTGTTACCGGGTGGGTCTTTACTATATTCATTCAAGTCCATTAATTCTCTTGATAAaggccactagtggatctg-3' (reverse). All gene disruptions were confirmed by Southern blot or PCR analysis.

Homozygous diploids deficient in three DNA damage resistance pathways were generated, or attempted to be generated, by mating viable haploid mutants or haploid mutant strains harboring pMK101 (a CEN-URA3 vector carrying RAD52, obtained from D. Livingston), pD293 (a CEN-ADE2 vector carrying RAD52), or pWS1506 (a CEN-URA3 vector carrying RAD1, obtained from W. Siede) followed by either nonselective or selective loss of the complementary plasmid (Ade^- or Ura^- segregants). Loss of the complementing plasmid and respective RAD locus was confirmed by PCR and MMS or UV sensitivity.

Diploids hemizygous at the MAT locus were generated by random disruption of one MAT allele with a PCR-generated mat::hph^R disruption fragment. Primers 5'-TTTTCGGGCTCATTCTTTCTTCTTTGCCAGAGGCTCACCGaggccactagtggatctg-3' (forward) and 5'-CCGCCACGACCACACTCTATAAGGCCAAATGTACAAACACcagctgaagcttcgtacg-3' (reverse) were used to amplify an 1.5-kb disruption fragment using pAG32 (GOLDSTEIN and MCCUSKER 1999 ) as template. The first 40 bases of each primer are complementary to the X and the Z1 sequences of MAT, respectively, and the 3' ends are complementary to the hygromycin B resistance cassette (lowercase letters). Following transformation, cells were plated on YEPD plates, incubated overnight, and replica plated onto YEPD plates containing hygromycin B. The mating type (and hence allele status at MAT) was assessed by mating transformants to appropriate mating-type testers.

Plasmid construction:
pD293 was generated by digestion of pMK101 (a CEN-URA3 vector carrying RAD52, obtained from D. Livingston) with EagI, followed by Klenow treatment to fill in the ends and subsequent digestion with SmaI. This results in removal of the URA3 gene and part of the tet gene. The large fragment was purified and ligated to a 3.6-kb BamHI/Klenow-treated fragment bearing the ADE2 gene (KAYTOR and LIVINGSTON 1996 ). The resulting CEN-ADE2 vector carrying RAD52 was confirmed by restriction digest and its ability to complement the MMS sensitivity of rad52 strains.

Tetrad analysis:
After incubation of diploid strains in sporulation medium for 5–7 days, cells were harvested by centrifugation and treated for 10 min at room temperature in predissection buffer (200 mM Tris·HCl, pH 9.0, 20 mM EDTA, 700 µM ß-mercaptoethanol; DAVIDOW and BYERS 1984 ). Cells were pelleted by centrifugation, resuspended in a 1/10 dilution of glusulase (Dupont NEN), and incubated at room temperature for 1 hr prior to dissection on YEPD. Dissected tetrads were allowed to germinate on YEPD for 2 days.

Viable spore colonies from all dissections were phenotypically screened for the presence of the appropriate DNA damage resistance markers. rev3::kan^R spore colonies and rad18::kan^R spore colonies were identified by replica plating onto YEPD containing Geneticin and scoring for growth after 1–2 days. rad52::URA3 or rad1::hisG spore colonies were identified by their MMS- or UV-sensitive phenotype, respectively. Ambiguous phenotypic results were confirmed by PCR.

Measurement of mutation rates:
Reversion rates of lys2Bgl and forward mutation rates at the CAN1 or ILV2/PDR1/SMR3 loci were determined by the method of the median (LEA and COULSON 1948 ). Independent 2-day-old colonies were inoculated into 5 ml of YEPD liquid medium and grown nonselectively to 2 x 10⁸ cells/ml on a roller drum. Cells were harvested by centrifugation, washed once with sterile water, and resuspended in 1 ml water. Aliquots (100 µl) of appropriate dilutions were plated onto SC-LYS to select Lys⁺ revertants, canavanine-containing medium to select forward mutations at the CAN1 locus, and sulfometuron-methyl-containing medium to select dominant forward mutations at the ILV2, PDR1, or SMR3 loci, and onto YEPD to determine viable cell numbers. Plating efficiency of a given strain was determined by dividing the average number of colony-forming units per culture by the average number of cells per culture (determined microscopically using a hemacytometer). Can^r colonies were counted on day 2, Lys⁺ colonies were counted on day 3, and SM^R colonies were counted on the earliest day detectable (day 2 or 3) after selective plating. The data from a minimum of 10 cultures were used for each rate determination. In all cases the median obtained was within the acceptable range for accurate rate calculations.

To eliminate outlying cultures (in terms of viable cell numbers) for rate determinations, the median culture population for each strain was determined among all cultures. This median was used to center a twofold acceptable range [median ± (median/3)] for individual culture populations, and outliers were excluded from further analysis. Contingency chi-square analysis was used to determine whether two rates were significantly different from each other (WIERDL et al. 1996 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Viability of haploids deficient in DNA damage resistance pathways:
We previously reported that haploid mutants deficient in BER (ntg1 ntg2 apn1 mutations) and either REC (rad52 mutation) or NER (rad1 mutation) were very slow growing (SWANSON et al. 1999 ). Haploid mutants deficient in BER and in the error-prone component of the PRR pathway, specifically DNA polymerase (TLS; rev3 mutation), exhibited no obvious growth defect (SWANSON et al. 1999 ). Attempts by transformation to directly construct strains deficient in BER and in any of the other two DNA damage resistance pathways were unsuccessful. In addition, an ntg1 ntg2 apn1 rad52 rev3 quintuple mutant bearing an extrachromosomal copy of RAD52 was unable to lose the plasmid spontaneously (data not shown), suggesting that a haploid mutant deficient for three DNA damage repair or bypass pathways might be inviable. To directly address this issue, diploid strains homozygous for one pathway and heterozygous for the remaining two pathways were constructed and then sporulated. In three of these diploids, both haploid parents were BER deficient (ntg1 ntg2 apn1) and each carried one additional dysfunctional pathway (Table 1). The TLS pathway, specifically the lesion bypass DNA Pol , was eliminated using the rev3::kan^R allele, resulting in resistance to Geneticin; the NER pathway was eliminated with the rad1::hisG allele, resulting in UV-radiation sensitivity; and the REC pathway was eliminated with the rad52::URA3 allele, resulting in uracil prototrophy and sensitivity to MMS. These diploids will be designated according to the haploid parents, each of which was BER^- and defective in one additional DNA damage resistance pathway (e.g., BER^-/TLS^- x BER^-/REC^-). In the fourth diploid, both haploid parents were TLS deficient (rev3::kan^R) and each additionally carried either the rad52::URA3 allele or the rad1::hisG allele. This diploid is referred to as TLS^-/REC^- x TLS^-/NER^-.

Meiotic tetrads derived from each diploid were dissected, and all viable spore colonies were assayed for the appropriate DNA damage resistance markers. The maximum viability that would be predicted if lethality results from the elimination of three pathways is 75%, assuming random segregation of the relevant mutant alleles. Representative dissection plates for each of the mutant crosses are pictured in Fig 1, and the viability patterns (alive:dead) are listed in Table 2. The BER^-/TLS^- x BER^-/REC^- cross resulted in 64% viability, the BER^-/TLS^- x BER^-/NER^- cross resulted in 62% viability, and the BER^-/REC^- x BER^-/NER^- cross resulted in 55% viability, all significantly lower than the wild-type cross viability (91%). The decreased viability in the mutant crosses is not due simply to the presence of a disrupted rad52 or rad1 allele, as control wild-type x rad52 and wild-type x rad1 crosses resulted in 91 and 88% viability, respectively (data not shown). When examined microscopically, 80% of the "dead" spores germinated, but formed microcolonies that failed to grow into visible colonies, even after extended periods of growth after dissection (data not shown). We estimate that these microcolonies represent <10 doublings. In contrast, the TLS^-/REC^- x TLS^-/NER^- cross resulted in 86% viability, suggesting that the TLS/REC/NER-deficient haploid is viable.

View larger version (76K): In this window In a new window Download PPT slide   Figure 1. Spore viability in dissected tetrads. (Left) Each column represents two dissected tetrads; the top four spore colonies were derived from one tetrad, and the bottom four spore colonies were derived from a second tetrad. (Right) Phenotypes of each viable spore colony with respect to Geneticin, MMS, or UV, as appropriate (S, sensitive; R, resistant). (A) BER-/TLS- x BER-/REC-. Geneticin sensitivity is given first (S, REV3; R, rev3) followed by MMS sensitivity (S, rad52; R, RAD52). A BER/TLS/REC-deficient spore colony would be designated "R,S." (B) BER-/TLS- x BER-/NER-. Geneticin sensitivity is given first, followed by UV sensitivity (S, rad1; R, RAD1). A BER/TLS/NER-deficient spore colony would be designated "R,S." (C) BER-/REC- x BER-/NER-. UV sensitivity is given first, followed by MMS sensitivity. A BER/NER/REC-deficient spore colony would be designated "S,S." (D) TLS-/REC- x TLS-/NER-. UV sensitivity is given first, followed by MMS sensitivity. A TLS/NER/REC-deficient spore colony would be designated "S,S."

The segregation of the two heterozygous repair deficiencies in the above diploids was followed by replica plating spore colonies onto Geneticin-containing medium (rev3) and by scoring UV sensitivity (rad1) or MMS sensitivity (rad52). For the BER^-/TLS^- x BER^-/REC^-, BER^-/TLS^- x BER^-/NER^-, and BER^-/REC^- x BER^-/NER^- crosses, a viable spore colony simultaneously displaying the relevant two markers was never seen (Fig 1), indicating that neither the NER, nor the REC, nor the PRR pathway alone can support haploid survival in yeast. The small size of the dissected spore colonies from these crosses generally correlates with BER/NER or BER/REC deficiency as previously reported (SWANSON et al. 1999 ). In contrast to the above crosses, the TLS^-/REC^- x TLS^-/NER^- cross did result in viable spore colonies resistant to Geneticin and sensitive to both MMS and UV radiation (Fig 1). Out of the 70 viable spore colonies, 12 show dual sensitivity (TLS^-/NER^-/REC^-), equal to 17%, compared to the 25% that would be predicted assuming random spore death.

Although the ready identification of TLS^-/NER^-/REC^- spore colonies indicates that the BER pathway alone may be sufficient for haploid viability, the loss of Pol -dependent TLS activity only partially inactivates the PRR pathway. A complex of Rad6p with the Rad18p protein is thought to function as the key regulator of the PRR pathway, and elimination of either protein would be predicted to completely disable the pathway (reviewed in BROOMFIELD et al. 2001 ). To more rigorously address whether the BER pathway alone is sufficient for haploid growth, a rad18::kan^R allele was introduced into a NER^- haploid, and a NER^-/REC^- x NER^-/PRR^- diploid was constructed. Unexpectedly, the spore viability from this cross was only 39%, with only 7 of the 125 rad1 spore colonies also containing the rad18 allele. Inviability of rad1-1 rad18-2 spores was not seen in an earlier study (DOWLING et al. 1985 ), and we speculate that the inviability we observe could be due to differences in strain backgrounds or to the use of deletion alleles. In spite of the low viability of NER^-/PRR^- spores, one of the 7 viable spore colonies was also REC^-, indicating that it is indeed possible for haploids to survive with only the BER repair pathway intact.

Spontaneous mutation rates of haploid DNA damage resistance mutants:
Since dissection analysis revealed that the NER/TLS/REC-deficient haploid is viable, isogenic haploid strains were generated by transformation and spontaneous mutation rates were determined using the lys2Bgl frameshift reversion assay (GREENE and JINKS-ROBERTSON 1997 ) and forward mutation at CAN1 (WHELAN et al. 1979 ). We previously reported synergistic increases in mutation rates in haploid strains deficient in BER and either NER (60- to 110-fold) or REC (40- to 150-fold); an NER-deficient, REC-deficient, or BER-deficient strain had an 1.8, 7-, or 5-fold, respectively, increase in mutation rate (SWANSON et al. 1999 ). Simultaneous loss of NER and REC likewise results in a significant 15- to 30-fold increase in mutation rate, consistent with a synergistic relationship at both lys2Bgl and CAN1 (Table 3). The effect of TLS mediated by Pol is revealed by an 30% decrease in frameshifts upon the loss of TLS in a NER-deficient strain. In the absence of REC, however, Pol -mediated TLS is the major contributor to frameshifts, with loss of Pol resulting in an 80% decrease in mutation rates. For the CAN1 assay, TLS is the major contributor to mutagenesis in both the NER- and REC-deficient backgrounds, accounting for 60–80% of the forward mutation events. Finally, loss of TLS in combination with both NER and REC decreases the spontaneous mutation rate 60% in the frameshift assay and 85% in the CAN1 assay. The genetic interactions observed here between the multiple DNA damage resistance pathways for spontaneous mutagenesis correlate well with those involved in radiation- or chemical-induced mutagenesis (LAWRENCE and CHRISTENSEN 1976 ; MCKEE and LAWRENCE 1980 ; TORRES-RAMOS et al. 2000 ; GELLON et al. 2001 ). These data support the role of TLS acting in competition with or being able to substitute for NER and REC in the resistance to a common spontaneous lesion(s). The decreased plating efficiency (16% of the wild-type plating efficiency) and the persistence of a significantly elevated mutation rate in the NER/TLS/REC-deficient haploid suggests that while the BER pathway has a high capacity and broad substrate specificity to repair spontaneous lesions, it has difficulty managing the entire load of spontaneous DNA damage.

Viability of homozygous diploids deficient in DNA damage resistance pathways:
To further address the roles of DNA damage resistance pathways in viability, homozygous diploid strains were generated. NER/TLS/REC-deficient haploids were mated directly to generate the corresponding diploid. To generate the other diploids, MATa and MAT strains deficient in BER and in two of the remaining three pathways (NER, REC, or TLS), but harboring a complementing plasmid for either NER (pWS1506) or REC (pMK101 or pD293) as appropriate, were constructed. After mating, counterselection against the complementing plasmid was attempted. Only after extended growth on 5-FOA were homozygous diploids deficient in BER/NER/TLS or BER/TLS/REC recovered. Furthermore, the BER/NER/TLS- and BER/TLS/REC-deficient diploids show reduced plating efficiencies relative to a wild-type control diploid. While the single and double DNA damage resistance pathway mutants show little, if any, decrease in plating efficiency, the BER/NER/TLS-, BER/TLS/REC-, and NER/TLS/REC-deficient diploids have 38, 13, and 18% plating efficiencies compared to a wild-type control diploid, respectively. Interestingly, the NER/TLS/REC-deficient diploid has a plating efficiency comparable to the respective haploid strain (16% of the wild-type haploid plating efficiency). A BER/NER/REC-deficient homozygous diploid was not obtainable using plasmid shuffle techniques (DSC183 x DSC184) over 130 generations of nonselective growth. Furthermore, DSC183 x DSC184 diploid colonies grown under nonselective conditions showed a mixed morphology of smooth and rough colonies and had a plating efficiency of <10% compared to a control wild-type diploid (data not shown). These observations suggest that simultaneous deficiency of BER/NER/REC does not support diploid growth.

Diploids differ from haploids in two significant ways, and either way might account for the haploid-diploid survival differences observed here. In addition to the obvious ploidy difference, haploids contain either the MATa or the MAT allele, whereas diploids contain both MAT alleles. Because heterozygosity at MAT has been shown to regulate use of the REC pathway (SCHILD 1995 ; MORGAN et al. 2002 ), we deleted one copy of the MAT locus in the two triply defective diploids described above that were able to lose the complementing plasmid (i.e., the BER/NER/TLS- and BER/TLS/REC-defective diploids containing the NER- and REC-complementing plasmids, respectively). The resulting mating-proficient diploids were also able to lose the complementing plasmids and to form colonies on 5-FOA (data not shown). We thus conclude that the haploid-diploid viability difference results from a ploidy difference rather than from heterozygosity at MAT.

Spontaneous mutation rates of homozygous diploid DNA damage resistance mutants:
SM is a sulfonylurea herbicide that acts as an inhibitor of the product of the ILV2 gene, acetolactate synthase (ALS; FALCO et al. 1985 ). ALS is a mitochondrial enzyme involved in the biosynthesis of the branched-chain amino acids isoleucine and valine (FALCO et al. 1985 ). The vast majority of spontaneous SM-resistant mutants act in a dominant fashion, with most of these mapping to the ILV2 locus and additional mutations mapping to the PDR1 and SMR3 loci. Semidominant and recessive mutations have also been identified, which also map to all three loci (FALCO and DUMAS 1985 ). The prevalence of dominant mutations among spontaneous SM-resistant mutants makes SM an ideal selective agent to assay spontaneous mutation rates and the impact of DNA damage resistance pathways on mutagenesis in diploids.

Using SM resistance as an indicator, mutation rates in diploids were determined (Table 4). BER-deficient and REC-deficient diploids display significant increases in mutation rate (3.8- and 10-fold increase, respectively, relative to wild type), while an NER-deficient diploid does not show any significant change. As expected, a TLS-deficient diploid has a decreased spontaneous mutation rate. When multiple deficiencies are combined, a greater than additive increase in mutation rate generally results. A BER/NER-deficient diploid shows a 110-fold synergistic increase in mutation rate compared to 3.8- and 1.4-fold, respectively, in the single mutants. Likewise, combining a REC deficiency, which results in a 10-fold mutation rate increase by itself, with a BER deficiency results in a 55-fold increase in the spontaneous mutation rate. Loss of TLS in a BER-deficient or NER-deficient strain decreases the spontaneous mutation rate 35 and 50%, respectively. The greatest contribution of TLS by Pol to spontaneous mutagenesis is observed when Pol is eliminated in REC-, BER/NER-, or BER/REC-deficient strains. The data indicate that Pol -mediated TLS accounts for 95% of the events in these backgrounds.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

There is a long history of examining the contributions of DNA damage resistance pathways to the repair of induced DNA damage in yeast. Early analyses focused on the responses of the RAD3, RAD52, and RAD6 epistasis groups in resistance to UV and -radiation or to chemical mutagens (reviewed in COX and GAME 1974 ; MOORE 1978 ; RUHLAND and BRENDEL 1979 ; MCKEE and LAWRENCE 1980 ; HENRIQUES and MOUSTACCHI 1981 ; PRAKASH and HIGGINS 1982 ). More recent studies have examined base lesions and DNA strand crosslinks introduced by chemical mutagens and also have considered the role of the BER pathway in the resistance to DNA damage in yeast (XIAO and CHOW 1998 ; SCOTT et al. 1999 ; TORRES-RAMOS et al. 2000 ; GELLON et al. 2001 ; GROSSMANN et al. 2001 ). Similar studies have been carried out in Schizosaccharomyces pombe and Escherichia coli, and, as in Saccharomyces cerevisiae, indicate overlapping specificities of the damage resistance pathways (ASAD et al. 2000 ; KUIPERS et al. 2000 ; MEMISOGLU and SAMSON 2000B ; OTTERLEI et al. 2000 ).

In contrast to induced DNA damage, studies examining contributions of the various DNA damage resistance pathways to spontaneous DNA damage resistance are far less prevalent in the literature. Our previous work examining the specificities of the DNA damage excision pathways (BER and NER) and the damage bypass pathways (specifically Pol -mediated TLS and REC) indicated that these pathways compete for the repair/bypass of a common spontaneous lesion(s). The work also documented poor growth of the BER/NER-deficient and the BER/REC-deficient strains (SWANSON et al. 1999 ). Attempts by transformation to construct strains simultaneously deficient in any three of the pathways were unsuccessful, suggesting that such mutant combinations may be lethal. These observations led us to examine the meiotic products of appropriate crosses for viable spore colonies deficient in three of the four pathways. Crosses thus were designed to examine simultaneous elimination of BER/TLS/REC, BER/NER/TLS, BER/NER/REC, and NER/TLS/REC. While BER has been severely compromised in the apn1 ntg1 ntg2 mutants used in the current studies, some level of BER activity is still likely to be present in the cells, including the abasic site (AP) endonuclease activity of Apn2p (SANDER and RAMOTAR 1997 ; BENNETT 1999 ; UNK et al. 2000 ) and the N-glycosylase and AP lyase activities of Ogg1p (VAN DER KEMP et al. 1996 ; THOMAS et al. 1997 ). Even though residual BER activity was likely in our BER-deficient strains, the elimination of BER and any two of the remaining three pathways was lethal in haploids. To our knowledge, this is the first report of a requirement for spontaneous DNA damage resistance pathways for viability in yeast when the cell metabolism is otherwise normal. Although we believe that the lethalities observed here result from the simultaneous deficiencies in the respective DNA damage resistance pathways, there are examples of synthetic lethality that result from specific allele combinations (MONTELONE et al. 1988 ; SONG et al. 1990 ; SYMINGTON 1998 ; KLEIN 2001 ).

While neither the NER, nor the REC, nor the PRR pathway alone was able to support haploid growth, the viability of the NER/TLS/REC-deficient strain suggested that the BER pathway alone may be sufficient for haploid survival. Because the Pol deficiency created in our strains did not completely eliminate lesion bypass capability, it was important to examine the broader role of PRR in the observed haploid survival. The RAD6 PRR pathway consists of at least three subpathways, one error-prone (Pol ) and two error-free subpathways (RAD5 and POL30/POL3; reviewed in BROOMFIELD et al. 2001 ). As a result, our TLS strains were compromised only in the error-prone component of PRR, with both error-free subpathways remaining fully functional, as well as the damage bypass activity of DNA polymerase . The Rad6 protein acts in concert with Rad18p to regulate all the mutagenic and error-free subpathways of the PRR pathway. While Rad18p appears to have roles only in PRR, Rad6p is required for a variety of biological processes in addition to DNA repair, including gene silencing, protein degradation, sporulation, and histone ubiquitination (LAWRENCE 1994 ). To completely disable PRR in addition to the NER and REC pathways, we thus attempted to obtain a rad1 rad52 rad18 triple mutant haploid by dissection of an appropriate diploid. The identification of an NER/PRR/REC-deficient spore colony in this analysis supports the conclusion that BER alone can support haploid growth. We note that the viability of the haploid NER/TLS/REC-deficient mutant is consistent with previous studies using alleles in the respective pathways that may not have been nulls (reviewed in COX and GAME 1974 ) and with studies in which only some of the alleles were nulls (reviewed in GAME 2000 ). The viability of the haploid possessing functional BER alone indicates that BER is the most critical of the DNA damage resistance pathways in yeast and may be responsible for the removal of either the majority of spontaneously occurring DNA damage or specifically those damages that are potentially lethal (Fig 2A). We note that residual error-prone PRR activity via a putative Rad5p/Rad6p pathway (CEJKA et al. 2001 ) and fully functional nonhomologous end-joining (reviewed in PASTINK et al. 2001 ) and mismatch repair (reviewed in MARTI et al. 2002 ) are still present in our repair-deficient backgrounds and may contribute to the survival of the NER/PRR/REC-deficient mutant.

View larger version (16K): In this window In a new window Download PPT slide   Figure 2. Spontaneous DNA damage resistance pathway hierarchies in haploids and diploids. (A) In haploids, BER has a central role in the resistance to spontaneous lesion "X," with NER, TLS, and REC serving as alternative pathways. If BER is eliminated, at least two other pathways must remain intact to compensate for the decreased repair capacity and still support growth. (B) In diploids, the hierarchy shifts to rely more heavily on REC and NER for the resistance to spontaneous DNA lesions. All the pathways except TLS can compensate for the loss of the remaining three resistance pathways and support diploid growth. Arrow thickness indicates proposed capacities for handling spontaneous DNA damage.

While simultaneous deficiency in BER/TLS/REC, BER/NER/TLS, or BER/NER/REC resulted in inviability in haploids, only the BER/NER/REC-deficient strain appeared inviable as a diploid. The viabilities of the BER/TLS/REC- and BER/NER/TLS-deficient diploids were unaffected by the status of the mating-type locus, indicating that the viability results from ploidy, rather than from mating-type heterozygosity, which has been implicated in suppression of some DNA repair defects (MARTIN et al. 1981 ; HEUDE and FABRE 1993 ; SCHILD 1995 ; ASTROM et al. 1999 ; MORGAN et al. 2002 ). The BER/TLS/REC-deficient, BER/NER/TLS-deficient, and NER/TLS/REC-deficient diploids, although viable, showed reduced plating efficiency. Furthermore, there appeared to be a hierarchy of plating efficiency, with functional REC alone reducing plating efficiency by only 60% relative to wild type, and functional BER or NER alone reducing plating efficiency by 85% relative to wild type. This hierarchal response may indicate that in the diploid there may be different priorities in the utilization of a given DNA damage resistance pathway compared to haploids (Fig 2B). The presence of a second genomic complement in a diploid and the higher plating efficiency of the diploid containing only functional REC may indicate that REC repairs collapsed replication forks at the sites of DNA damage (KUZMINOV 2001 ), or it may point to the reliance on the second copy of a locus for the repair of the damaged copy, as previously reported for UV- and -ray-induced mutagenesis (RAO and REDDY 1982 ; HEUDE and FABRE 1993 ). As a second copy is present only in the late S/G2 phase of the cell cycle in haploids, this might explain the lethality of the BER/NER/TLS-deficient combination in haploids.

Haploid yeast mutants deficient in the proofreading activities of the DNA polymerases and or in the proofreading activity of Pol and the mismatch repair machinery are inviable, while the corresponding diploids are viable (MORRISON et al. 1993 ; MORRISON and SUGINO 1994 ). In these mutants, the viability of diploids has been attributed to their ability to tolerate a much higher mutational load than haploids due to the presence of a second genomic complement (but see also DATTA et al. 2000 ). The cause of the haploid lethality reported here may reflect a similar error "catastrophe," or accumulation, as simply unrepaired lesions or unresolved recombination intermediates blocking DNA replication would be expected to cause lethality in the homozygous diploid as well as in the haploid background. Interestingly, diploids possessing functional PRR alone were not identified, suggesting that error accumulation and/or replication-blocking lesions may be causing death in these cells. While the mutagenic nature of the TLS DNA polymerase and the viability of diploids possessing only functional BER or NER point to the former possibility, the latter may occur if the PRR proteins have already reached saturation, and the excess lesions result in the blocking of DNA polymerase. These blocks may otherwise be repaired by REC, but in its absence S-phase arrest may be permanent, causing death.

Because mutations at CAN1 are recessive, this locus could not be used for mutation rate analysis in diploids (WHELAN et al. 1979 ). Spontaneous forward mutation to SM^R, however, provides a convenient way to measure forward mutation in diploids due to the predominance of dominant mutations, which map primarily to ILV2. The diploid forward mutation rate to SM^R in the current study closely parallels previous Can^r forward rates observed in haploids (compare Table 3 and Table 4 in this article with Table 1 of SWANSON et al. 1999 ). The characterized enzyme kinetics of spontaneous SM^R mutants (FALCO and DUMAS 1985 ) suggest that SM^R mutants, like Can^r mutants (TISHKOFF et al. 1997 ), are predominantly a consequence of base substitution mutations in a wild-type background. Furthermore, it is likely that in the mutant backgrounds similar events occur in both the CAN1 and sulfometuron methyl assay. Using SM^R as a selective marker in diploids, it is evident that, as in haploids, the DNA damage resistance pathways BER, NER, TLS, and REC have synergistic relationships and are able to compensate for each other in the resistance to spontaneously occurring DNA lesions. The observations here and previously (SWANSON et al. 1999 ) correlate well with those reported in radiation- or chemical-induced mutagenesis studies (LAWRENCE and CHRISTENSEN 1976 ; MCKEE and LAWRENCE 1980 ; TORRES-RAMOS et al. 2000 ; GELLON et al. 2001 ).

Spontaneous DNA lesions encompass a wide array of base damages, including oxidative DNA damage, which is thought to be processed predominantly by BER. The observed appearance of small colonies or mixed colony morphology in some of our repair backgrounds may indicate petite formation as a counteractive mechanism to enhance survival. While previous experiments in isogenic repair backgrounds showed little or no significant effect of anaerobic growth (MOREY 1999 ), further analysis of the various repair-deficient backgrounds under anaerobic or oxidative stress conditions could further elucidate the significance of oxidative lesions as a commonly recognized lesion by the BER, NER, REC, and TLS pathways. Given the reported redundancy for the resistance to abasic sites in E. coli and yeast (SWANSON et al. 1999 ; OTTERLEI et al. 2000 ; TORRES-RAMOS et al. 2000 ; GELLON et al. 2001 ), it seems likely that at least one of the common lesion(s) is an abasic site. In vitro data indicate that the human NER machinery can remove abasic sites (HUANG et al. 1994 ) and that the yeast TLS Pol can bypass these sites (NELSON et al. 1996 ; HARACSKA et al. 2001 ), lending further support for the role of abasic sites as a common lesion.

The results reported here demonstrate the critical role of DNA damage resistance pathways in the viability and genome stability of both haploid and diploid yeast strains. Furthermore, the hierarchy of DNA damage resistance appears to be different on the basis of ploidy, with haploids being more dependent on BER and diploids more dependent on REC for survival. Repair mechanisms appear to be more critical in the haploid state, where each essential gene must be preserved to permit survival, while in the diploid state repair can occur with delayed timing as the second genome can complement the damaged locus. The increased dependence on recombination in the diploid further allows the advantageous use of the second genome copy to help repair a damaged locus at any stage of the cell cycle. The absolute requirement in yeast for some minimal capacity to repair or tolerate DNA damage is likely a characteristic of other organisms. The hierarchy of DNA damage resistance pathways may likewise be conserved, with haploid organisms relying more heavily on damage removal (BER and NER) and diploid or polyploid organisms shifting to damage tolerance mechanisms (TLS and REC). Spontaneous DNA damage is an unavoidable consequence of cellular metabolism, and these data reveal a threshold requirement of damage removal and/or mutation avoidance to sustain life.

	FOOTNOTES

¹ Present address: HIV and Immunology Diagnostics Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.

	ACKNOWLEDGMENTS

We thank DuPont for their generous donation of the sulfometuron methyl used in this work and John Game for his helpful comments on the manuscript. S. Jinks-Robertson was supported by National Institutes of Health (NIH) grant GM-38464. P. W. Doetsch was supported by NIH grants CA-78622 and CA-73401. N. J. Morey was partially supported by the Graduate Division of Biological and Biomedical Sciences of Emory University.

Manuscript received April 5, 2002; Accepted for publication February 19, 2003.

	LITERATURE CITED

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