INTRODUCTION

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DNA alkylation damage is repaired by the base excision repair (BER) pathway in a variety of organisms (12). During BER, the DNA lesion is first recognized and removed from the sugar-phosphate backbone by a DNA glycosylase, creating an abasic site. This abasic site is subsequently processed by an apurinic/apyrimidinic (AP) endonuclease and an exonuclease. The resulting small gap is filled in by a DNA polymerase, and the nick is sealed by a DNA ligase (12). The biochemical characteristics of DNA polymerase  (Pol ) indicate that it is the polymerase that fills in the resulting gap. Pol  has high affinity for duplex DNA containing small gaps (30, 33). Pol  is processive when filling small gaps of up to 6 nucleotides (30) and has a higher catalytic efficiency on 5' phosphorylated single-base-pair gaps (6), and purified Pol  protein functions in BER in vitro (15, 16, 31). Evidence from numerous laboratories has implicated Pol  as playing a role in DNA repair in vivo. For example, chemical inhibitors of Pol  inhibit DNA repair in cells treated with DNA-damaging agents such as methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), bleomycin, and  irradiation (11, 14, 19). Simian virus 40-transformed mouse fibroblasts that lack Pol  are more sensitive to MMS than are their wild-type counterparts (31). Finally, in addition to its DNA polymerase activity, Pol  is able to catalyze the removal from DNA of 5'-deoxyribose-phosphate termini that sometimes result from the action of an AP endonuclease (18). This also provides evidence that Pol  participates in BER.

In Saccharomyces cerevisiae, a putative homolog of mammalian DNA Pol  has been identified. The polymerase IV gene encodes a protein with 26% identity and 50% similarity to Pol  (4). The biochemical characteristics of Pol IV are very similar to those of mammalian DNA Pol  (23, 29). Disruption of the Pol IV gene does not result in a significantly different phenotype from that observed in Pol IV⁺ strains with respect to DNA repair, growth, and sporulation (17, 23); this could be due to functional redundancy in S. cerevisiae. We previously demonstrated that S. cerevisiae cells expressing dominant negative mutants of rat Pol , a putative Pol IV homolog, are sensitive to MMS and not to UV light (10). At that time, we suggested that the dominant negative rat Pol  mutants interfered with BER in S. cerevisiae. Recently, Bhattacharyya and Banerjee showed that a variant of Pol  associated with colon cancer also acts as a dominant negative mutant (2).

Three genes in S. cerevisiae that are known to specifically repair DNA alkylation damage have been cloned. The 3-methyladenine DNA glycosylase gene product (MAG) releases 3-methyladenine (MeA) and 7-methylguanine (7MeG) bases from DNA (7-9). The APN1 gene encodes the major AP endonuclease of S. cerevisiae (22, 24) and is responsible for processing the abasic sites that remain after the action of MAG. Both of these proteins function in BER. The O⁶-methylguanine DNA methyltransferase (MGT) is encoded by the MGT1 gene in S. cerevisiae (26, 35, 36). This protein repairs alkylation damage by directly transferring the methyl group from the methylated base to a cysteine residue on the MGT protein itself (26, 27). Each of these enzymes functions to protect the cell from endogenous alkylation damage resulting in a low rate of spontaneous mutagenesis in S. cerevisiae (37).

To determine if the rat Pol  dominant negative mutants interfere with BER specifically, we characterized the functional relationship between our rat DNA Pol  dominant negative mutants with the MAG, APN1, and MGT1 gene products. Our results demonstrate that the DNA polymerase  mutants specifically interfere with the gap-filling step of BER and suggest that mutagenesis can occur during gap filling in vivo.

	MATERIALS AND METHODS

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Yeast strains and media. RS1 (MATa his3 leu2 ura3-52 trp1-289a GAL⁺) and RS8 (MATa his3 leu2 ura3-52 trp1-289a GAL⁺) were obtained from L. Samson (Harvard School of Public Health). Both the apn1-1::HIS 3 and the mag1-2::LEU2 strains were obtained from L. Samson (37) and were created in the RS8 background. The mgt1-1::LEU2 strain was created as described previously (35) by disrupting the endogenous MGT1 gene in RS1 with the mgt1::LEU2 in vitro cassette. Disruption at the MGT1 locus was verified by Southern hybridization (data not shown) (25) and sensitivity to MNNG (see below). Rich medium (YPD) was prepared as described previously (28). Synthetic complete (SC) medium (28) was supplemented with 2% raffinose instead of glucose and with the appropriate amino acids. Transformants of all strains were selected on SC medium-2% raffinose in the absence of uracil (for pYES2 vector).

Plasmids. Yeast expression plasmids for pBK3 (Pol -WT-expressing plasmid), pPol -TR, and pPol -14 were constructed as described previously (10) in pYES 2 with the URA3 selectable marker for transformation.

Cell survival. Survival data were obtained by a modification of previously described methods (10). Briefly, cells were grown overnight in YPD-2% glucose. Cells from the overnight culture were then washed three times with sterile water and used to inoculate (1:50 from overnight culture) SC medium or SC medium lacking the appropriate amino acid for plasmid selection plus 2% raffinose to suppress or 2% galactose to induce Pol  protein expression and incubated at 30°C overnight to a cell density of 1 × 10⁷ to 2 × 10⁷ cells/ml. The cells were treated with 0.3% MMS or 30 µg of MNNG per ml at 30°C. Aliquots were removed at various times, diluted into 0.05 M potassium phosphate buffer (pH 7.0), and plated on YPD. The cells were incubated for 3 days at 30°C, and colonies were counted.

Mutagenesis. Spontaneous mutation rates for Trp⁺ reversion were calculated by using fluctuation analysis by a modification of the method of von Borstel (32). Briefly, cells were grown overnight in YPD and washed three times with sterile water. These cells were used to inoculate (1:100) SC medium lacking uracil for plasmid selection and containing 2% raffinose. The cells were grown to 10⁷ cells/ml, diluted 1:10 into fresh medium, and grown for 3 h at 30°C. The culture was induced to express Pol  protein by further incubation with 0.5% galactose for 3 h at 30°C. Then 4 × 10³ cells per ml were grown to 1 × 10⁷ cells per ml (12 days at 30°C) in 240 individual 1-ml cultures in SC medium with limiting tryptophan (1.5 µM). The number of wells with no Trp⁺ revertants after the incubation period (12 to 14 days at 30°C) was used to calculate the mutation rate (32).

	RESULTS

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To study the in vivo role of a protein, one approach is to disrupt its normal cellular function and characterize the resulting phenotype. Because disruption of both copies of the Pol  gene in mice results in death (13) and because disruption of the Pol IV gene in S. cerevisiae does not result in any significant cellular phenotype (17, 23), gene disruption studies have been inconclusive. Therefore, to begin to study the cellular role of Pol  and its S. cerevisiae Pol IV homolog, we constructed rat Pol  dominant negative mutants (10). One of these mutants, Pol -TR, contains the first 170 amino acids of rat Pol  and has no catalytic activity. The other mutant, Pol -14, which contains a point mutation that alters amino acid residue 265 from cysteine to tyrosine, has catalytic activity similar to that of wild-type Pol  (34). We previously demonstrated that expression of the rat DNA Pol  dominant negative mutants in S. cerevisiae resulted in cellular sensitivity specifically to the DNA alkylation agent MMS but not to UV light (10). These data suggested that Pol  dominant negative mutants interfere with BER and not nucleotide excision repair (NER) (10). However, because DNA alkylation damage is not repaired exclusively by the BER pathway, in this report we characterize the epistatic relationship of our dominant negative mutants to the functions of known enzymes of the BER pathway in S. cerevisiae.

DNA Pol  mutants are epistatic to the APN1 and MAG gene products. We compared the sensitivity of the wild-type, apn1-1::HIS3, and mag1-2::LEU2 strains to MMS in the presence and absence of Pol -TR and Pol -14 to determine if these proteins interfere with BER in S. cerevisiae. Figure 1 shows the MMS survival curves of the various strains with and without expression of Pol -TR (Fig. 1A and C) or Pol -14 (Fig. 1B and D). When expressed in the wild-type background Pol -TR (Fig. 1A) and Pol -14 (Fig. 1B), mutant proteins sensitize the wild-type RS8 strain to MMS. The sensitizing effect of expression of Pol -14 or Pol -TR in the RS8 genetic background is limited to the shoulder portion of the curve. We offer three explanations for this phenomenon. The most likely explanation is that due to the heterologous nature of the system and the methods we used to express the proteins, expression is not adequate or does not occur long enough to alter the rate of cell killing (i.e., to alter the slope). Second, we suggest that the proteins are turned over rapidly and are not present long enough to significantly alter the rate of cell killing. Third, the Pol -TR and Pol -14 proteins are derived from the rat Pol  enzyme and may not interact with proteins in S. cerevisiae that function to repair DNA damage. A lack of interaction with other BER proteins may not allow Pol  to gain access to sites in DNA in need of repair or to participate in a repair complex of proteins. We observed no increase in MMS sensitivity when we expressed the Pol -TR or Pol -14 proteins in either the apn-1-1::HIS3 (Fig. 1A and B) or the mag1-2::LEU2 (Fig. 1C and D) background. This result demonstrates that the Pol  mutant proteins are epistatic to the APN1 and MAG1 gene products and therefore most probably function in the same pathway. Since it is known that MAG and APN1 act sequentially in the BER pathway of yeast, our data suggest that the rat Pol  dominant negative mutants interfere with BER downstream of the action of APN1, most probably during the gap-filling step of BER.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Epistasis analysis of Pol  mutants with APN1 and MAG. Yeast strains expressing Pol -TR (A and C) or Pol -14 (B and D) were grown and induced to express Pol  protein as described in Materials and Methods. (A) MMS sensitivity of wild-type (open squares), Pol -TR in wild-type (solid squares), apn1-1::HIS3 (open circles), and Pol -TR in apn1-1::HIS3 (solid circles) strains. (B) MMS sensitivity of wild-type (open triangles), Pol -14 in wild-type (solid triangles), apn1-1::HIS3 (open diamonds), and Pol -14 in apn1-1::HIS3 (solid diamonds) strains. (C) MMS sensitivity of wild-type (open squares), Pol -TR in wild-type (solid squares), mag1-2::LEU2 (open circles), and Pol -TR in mag1-2::LEU2 (solid circles) strains. (D) MMS sensitivity of wild-type (open triangles), Pol -14 in wild-type (solid triangles), mag1-2::LEU2 (open diamonds), and Pol -14 in mag1-2::LEU2 (solid diamonds) strains. S. cerevisiae strains harboring the vector alone or a nondominant mutant, when induced with galactose to express protein, showed no increase in MMS sensitivity (reference 10 and data not shown). The results are representative of three independent determinations; all determinations demonstrated similar results.

Pol  mutants are epistatically distinct from the MGT1 gene product. Since alkylation damage can also be repaired via a methyltransferase pathway, a pathway independent from BER in S. cerevisiae, the specificity of the Pol  mutants was assayed by investigating the epistatic relationship of Pol -TR and Pol -14 to the O⁶-methylguanine DNA methyltransferase (MGT1) gene product. Figure 2 shows the MNNG survival curves of S. cerevisiae wild-type and mgt1-1::LEU2 strains with and without expression of the rat Pol  mutant proteins. Expression of Pol -TR (Fig. 2A), or Pol -14 (Fig. 2B) sensitizes the wild-type strain to MNNG. The mgt1-1::LEU2 strain is very sensitive to MNNG, as reported previously (36, 37). However, expression of either Pol -TR (Fig. 2A) or Pol -14 (Fig. 2B) in the mgt1-1::LEU2 strain further sensitizes this strain to MNNG. These data demonstrate that the Pol  mutant proteins do not interfere with the methyltransferase pathway of DNA alkylation damage repair.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Epistasis analysis of Pol  mutants with MGT1. Yeast cells expressing Pol -TR (A) or Pol -14 (B) were grown and induced to express protein as described in Materials and Methods. (A) MNNG sensitivity of wild-type (open squares), Pol -TR in wild-type (solid squares), mgt1::LEU2 (open circles), and Pol -TR in mgt1::LEU2 (solid circles) strains. (B) MNNG sensitivity of wild-type (open triangles), Pol -14 in wild-type (solid triangles), mgt1::LEU2 (open diamonds), and Pol -14 in mgt1::LEU2 (solid diamonds) strains. The results are representative of two independent determinations; all determinations demonstrated similar results.

Pol -14 increases the spontaneous mutagenesis rate in yeast. The above results demonstrate that expression of rat Pol  dominant negative mutants specifically interferes with BER in S. cerevisiae. The BER pathway is a major DNA repair pathway in S. cerevisiae for the repair of alkylation damage. Alkylation damage results from both exogenous and endogenous sources, and it has been demonstrated previously that inhibiting BER in S. cerevisiae leads to an increase in the spontaneous mutation rate (37). Therefore, enzymes that play a role in this pathway are responsible for maintaining and monitoring the genomic fidelity of the cells. To determine if expression of the dominant negative mutants results in an increase in spontaneous mutagenesis, we measured the spontaneous rate of reversion to Trp⁺ by using the auxotrophic allele trp1-289 in strains overexpressing either Pol -WT, Pol -TR, or Pol -14. The trp 1-289 allele is the same allele as that used by Xiao and Samson (37). This allele is an amber mutation that can revert to Trp⁺ directly at the site of the mutation or by amber suppressor mutations, thus allowing us to measure a variety of in vivo mutational events. Figure 3 shows the fold increase of the spontaneous mutation rate over the RS8 strain in the presence of either the Pol -WT, Pol -TR, or Pol -14 protein. We observed no increase over RS8 in the spontaneous mutation rate when the Pol -WT or Pol -TR was expressed and a fivefold increase when the Pol -14 protein was expressed. We observed a 10-fold increase over that for Pol -WT when we expressed Pol -14 in the RS8 strain. This increase in the spontaneous mutation rate is similar to the increase observed in cells deficient in APN1 (37). This effect is specific for the expression of the Pol -14 protein, since the same strain gave no increase in the mutation rate in the absence of the inducing agent (data not shown). These results suggest that interference with BER does not always result in an increase in mutagenesis.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Pol -14 increases the spontaneous mutation rate in S. cerevisiae. Strains were grown and induced to express Pol -14, Pol -TR, or Pol -WT protein as described in Materials and Methods. In the absence of the inducing agent, galactose, the spontaneous mutation rates of RS8 with either Pol -14, Pol -TR, or Pol -WT were within a range of 1 to 1.2 fold over that with RS8 alone.

Pol -14 makes mutations during the gap-filling step of BER in S. cerevisiae. To determine if Pol -14 functions downstream of the APN1 protein in mutagenesis, we measured the spontaneous mutation rate in the apn1-1::HIS3 strain expressing Pol -14, the protein that increased the spontaneous mutation rate of the RS8 wild-type strain. We reasoned that if gaps were required for mutagenesis in the presence of Pol -14, we would not see an increase in the spontaneous mutation rate in cells lacking APN1 activity. Figure 4 shows a comparison of the spontaneous mutation rate of the wild-type RS8 strain and apn1-1::HIS3 strain in the absence or presence of Pol -14 expression. As noted above, the background rate of Trp⁺ reversion in the wild-type RS8 strain is low (0.5 × 10⁸) and is increased fivefold in the presence of Pol -14 (2.5 × 10⁸). However, as shown in Fig. 4, expression of Pol -14 in the apn1-1::HIS3 background does not result in an increase in the spontaneous mutation rate compared with that of the apn1-1::HIS3 strain alone (compare the apn1-1::HIS3 strain alone [3.7 × 10⁸] with the apn1-1::HIS3 strain expressing Pol -14 [2.9 × 10⁸]). This result strongly suggests that the ability of Pol -14 to increase the spontaneous mutation rate is dependent on the activity of the APN1 gene product and most probably requires the presence of a gap.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Spontaneous mutation rate in the apn1-1::HIS3 strain expressing Pol -14. Yeast cells were grown and induced to express protein as described in Materials and Methods. Cells were diluted into minimal medium with limiting tryptophan (1.5 µM), and Trp+ revertants were scored as described in Materials and Methods. The results are the average of two independent determinations.

	DISCUSSION

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Previous work from our laboratory demonstrated that rat DNA Pol  dominant negative mutants sensitized wild-type S. cerevisiae cells to MMS but not to UV light. At that time, we suggested that our rat DNA Pol  dominant negative mutants interfered with BER but not NER in S. cerevisiae (10). We hypothesized that the mutant proteins disrupted BER by binding to the DNA nonproductively, preventing the gap from being filled in a timely manner, and ultimately resulting in an increase in the cytotoxicity of the yeast cells to MMS. These results prompted us to investigate the relationship of our mutant proteins to the BER pathway. This pathway has been well studied in S. cerevisiae, and the genes known to be involved in BER have been cloned and their functional relationships have been established (for example, see reference 37). The epistasis analysis we describe here with various S. cerevisiae strains that are disrupted in known BER proteins demonstrates that the DNA Pol  dominant negative mutants function in the same DNA repair pathway as the MAG and APN1 gene products with respect to repair of DNA alkylation damage (Fig. 1). However, the Pol  dominant negative mutants are not epistatic to the MGT1 gene product (Fig. 2), an enzyme known to function in a pathway distinct from BER. We conclude that the rat Pol -TR and Pol -14 mutants interfere specifically with BER in S. cerevisiae.

Pol -TR does not fill the gap. Pol -TR contains the DNA binding domain of the Pol  protein but lacks the aspartic acid residues that are essential for catalysis (5, 20). We suggest that Pol -TR competes with an endogenous S. cerevisiae DNA polymerase, perhaps Pol IV or Pol  (3), for binding to the gapped DNA. Because Pol -TR lacks the ability to catalyze DNA synthesis, the gaps to which it is bound remain unfilled, resulting in cell death.

Pol -14 fills gaps slowly. Pol -14 may also block the access of a gap to an endogenous S. cerevisiae DNA polymerase. However, Pol -14 is only about 10 times less active than wild-type Pol  (34) in vitro, indicating that it has a reduced ability to fill in gaps (31a). We suggest that in S. cerevisiae cells, Pol -14 most probably fills gaps slowly or fills fewer gaps in vivo, resulting in an increased sensitivity to MMS.

Pol -TR and Pol -14 do not act by inhibiting APN1. Recently, it has been shown that human DNA polymerase  interacts with human Ape, the major AP endonuclease in human cells (1). Therefore, it is possible that the Pol  mutants are acting by inhibiting APN1. We do not favor this explanation for the following reasons. First, if Pol -TR or Pol -14 were inhibiting the action of APN1, we would expect that expression of these proteins would sensitize the cells to the same extent as would result from disrupting the APN1 gene. This is not the case. Second, in the case of mutagenesis, whereas Pol -14 expression does result in a similar increase in the rate of spontaneous mutagenesis as that observed in APN1-containing strains, expression of Pol -TR has no effect on mutagenesis. If expression of Pol -TR inhibited the action of APN1, we would expect to observe an increase in spontaneous mutagenesis similar to that observed in APN1-disrupted strains. Finally, Bennett et al. demonstrated that APN1 stimulates the activity of Pol  by loading Pol  onto a gap (1). These data suggest that APN1 acts at a step prior to that of Pol . Therefore, mutants of Pol  should not decrease the activity of APN1 but, instead, should act after APN1.

Pol -TR and Pol -14 may interfere with Pol IV. Pol -TR and Pol -14 interfere with the gap-filling step of BER, suggesting that they interfere with the action of a DNA polymerase. The identity of the cellular polymerase whose function is inhibited by our dominant negative mutants remains to be uncovered. Pol -TR and Pol -14 may interfere with Pol IV. Pol IV has homology to Pol  and biochemical properties similar to Pol  (23, 29), an enzyme that has been implicated as functioning in BER. Therefore, by analogy, Pol IV may function in BER in S. cerevisiae. However, no significant cellular phenotype results from the disruption of the Pol IV gene (20, 23), suggesting either that Pol IV does not act in BER or that another polymerase substitutes for Pol IV when the cells are deficient in this enzyme. If Pol IV acts during BER, our dominant negative mutants would most probably block the action of this enzyme because the substrate specificities of Pol IV and Pol  are similar (23, 29).

Pol -TR and Pol -14 may interfere with Pol . Blank et al. suggested that Pol  functions in BER in S. cerevisiae because the Pol  mutant allele cdc2-2 is sensitive to alkylating agents (3). Therefore, Pol -TR and Pol -14 may bind to gaps that are normally filled in by Pol , interfering with the action of this enzyme. Alternatively, Pol -TR and Pol -14 may block the action of another polymerase in S. cerevisiae.

Pol -14 commits errors during the gap-filling step of BER. BER is a major cellular repair pathway that acts to repair both endogenous and exogenous alkylation damage. Inhibition of the early steps of BER has been shown to result in mutagenesis (37). Our present work extends these findings by focusing on the action of the DNA Pol and the consequences to the cell if BER malfunctions at the gap-filling stage. In our study, both Pol -TR and Pol -14 interfere with the gap-filling step of BER, as demonstrated by our epistasis analysis (Fig. 1). However, only expression of Pol -14 increases the spontaneous mutation rate (Fig. 3) in the wild-type strain to a level similar to that observed in an APN1-disrupted strain (37). Furthermore, the increase in the spontaneous mutation rate we observed in cells expressing Pol -14 is dependent upon APN1, because we did not observe an increase in the spontaneous mutation rate in cells expressing Pol -14 but lacking APN1. These data suggest that mutagenesis resulting from the expression of Pol -14 occurs after the abasic site is removed and that it most probably requires a gapped DNA substrate. Because we observed an increase in the spontaneous mutation rate only in the presence of Pol -14 and not Pol -TR, our data suggest that interfering with the gap-filling step of BER does not necessarily result in mutagenesis and indicates that gaps themselves are not mutagenic intermediates. Our results with the Pol -TR mutant demonstrate that it is not the loss of gap filling itself that is mutagenic, but the loss of correct gap filling that leads to mutations.