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DNA Polymerase 4 of Saccharomyces cerevisiae Is Important for Accurate Repair of Methyl-Methanesulfonate-Induced DNA Damage
http://www.100md.com 遗传学杂志 2006年第1期
     ABSTRACT The DNA polymerase 4 protein (Pol4) of Saccharomyces cerevisiae is a member of the X family of DNA polymerases whose closest human relative appears to be DNA polymerase . Results from previous genetic studies conflict over the role of Pol4 in vivo. Here we show that deletion of Pol4 in a diploid strain of the SK1 genetic background results in sensitivity to methyl methanesulfonate (MMS). However, deletion of Pol4 in other strain backgrounds and in haploid strains does not yield an observable phenotype. The MMS sensitivity of a Pol4-deficient strain can be rescued by deletion of YKu70. We also show that deletion of Pol4 results in a 6- to 14-fold increase in the MMS-induced mutation frequency and in a significant increase in AT-to-TA transversions. Our studies suggest that Pol4 is critical for accurate repair of DNA lesions induced by MMS.

    SACCHAROMYCES cerevisiae DNA polymerase 4 is a member of the Pol X family of DNA polymerases. The Pol X family is related to a larger group of nucleotidyltransferases (ARAVIND and KOONIN 1999) and includes polymerases ?, , μ, and the template-independent terminal deoxynucleotidyltransferase. Of these enzymes, Pol4 shares the most sequence homology with mammalian DNA polymerases and ?, respectively.

    S. cerevisiae DNA polymerase 4 is a 68-kDa protein first identified on the basis of sequence similarity to Pol? (BORK et al. 1992). The purified protein exhibits distributive DNA synthesis activity on a 3'-recessed primer template substrate and processive DNA synthesis on a small five-nucleotide gapped substrate (PRASAD et al. 1993; SHIMIZU et al. 1993). Deletion of the Pol4 gene demonstrated that it is not essential for viability, but there are conflicting results regarding its role in vivo. PRASAD et al. (1993) found no role for Pol4 in sporulation or spore viability, no alteration of spontaneous mutation frequencies in pol4 vs. wild-type strains, and no sensitivity of the pol4 strain to DNA-damaging agents alone or in combination with other polymerase mutants. BUDD and CAMPBELL (1995) showed that Pol4 does not function in repair of DNA damage but they reported that Pol4 is induced during meiosis and that pol4 spores had decreased viability and slow growth when compared to wild-type spores. Similar to the findings of BUDD and CAMPBELL, LEEM et al. (1994) reported that Pol4 is induced during meiosis but pol4 spores in their study had wild-type levels of viability. In addition, they found that pol4 cells had increased rates of illegitimate mating and that diploid cells had increased rates of meiotic intragenic recombination, were sensitive to methyl methanesulfonate (MMS), and exhibited elevated levels of meiotic double-strand breaks (DSBs) (LEEM et al. 1994). Therefore, unlike previous findings, LEEM et al. (1994) reported that Pol4 does function in the repair of MMS-induced DNA damage and showed that this function is in a pathway separate from that of Rev3.

    More recently, Pol4 has been found to play a role in end-joining reactions, involving a plasmid substrate, when end processing is required (WILSON and LIEBER 1999). Additionally, Pol4 is known to interact with S. cerevisiae Rad27 and Dnl4/Lif1 and to function in end-joining reactions in vitro in concert with these proteins (TSENG and TOMKINSON 2002, 2004).

    Interestingly, aside from the conflicting findings of LEEM et al. (1994) and BUDD and CAMPBELL (1995) regarding spore viability, all genetic experiments yielding a pol4 phenotype were performed in diploid strains of an SK1 background, suggesting that variation in strain background and genetic differences between SK1 and other wild-type laboratory strains (PRIMIG et al. 2000; WILLIAMS 2002) have an influence on Pol4 function.

    The findings of LEEM and colleagues, together with the known roles of Pol and Pol? in DNA repair and genome maintenance, prompted us to readdress the role of Pol4 in vivo. We found that Pol4-deficient cells are sensitive to MMS in an SK1 strain background and that the polymerase activity of Pol4 is important for the repair of MMS-induced DNA damage. Further examination of Pol4 by epistasis analysis with YKU70, which functions in nonhomologous end joining (NHEJ), suggests a role for Pol4 in NHEJ in vivo. In addition, we show that deletion of Pol4 does not affect spontaneous mutation frequency but it does result in a 6- to 14-fold increase in the MMS-induced mutation frequency. The types of mutations induced in MMS-treated cells lacking Pol4 appear to differ from those that arise in wild-type cells. Therefore, this work demonstrates a function for Pol4 in the maintenance of genomic stability during repair of MMS-induced alkylation damage and is consistent with previous findings of a role for Pol4 in the repair of DNA strand breaks through a NHEJ mechanism (WILSON and LIEBER 1999; TSENG and TOMKINSON 2002, 2004).

    MATERIALS AND METHODS

    Yeast media, strains, and genetic manipulations:

    Yeast strains were grown in standard media (GUTHRIE 1991). Three different strain backgrounds were employed in the experiments described here and are noted in Table 1. Cells were grown nonselectively in YPADU (1% yeast extract, 2% Bacto-peptone, 2% dextrose, supplemented with 0.3 mM adenine and 0.2 mM uracil, and 2% agar for plates) and selected growth was in synthetic complete (SC) media lacking the appropriate amino acids. SC media lacking arginine and containing 60 mg of canavanine per liter were used to identify forward mutations in the CAN1 gene. For study of the lys2Bgl allele frameshift assay, colonies were grown in YPEGE (1% yeast extract, 2% Bacto-peptone, 2.5%, supplemented with 2% glycerol/2% ethanol) and plated either on YPADU, for plating efficiency, or on media without lysine to detect frameshift lys2Bgl revertants (GREENE and JINKS-ROBERTSON 1997).

    The genotypes of strains used in this study are listed in Table 1. JSY1 and JSY2 were gifts of Akio Sugino. The pol4::URA strains were constructed using one-step targeted gene disruption. The deletion cassette was amplified by PCR with the primers CHS5 (5'-atcttttaatgattattaagatttttattaaaaaaaaattcgctcctcttgggtaataactgatataatt-3') and CHS6 (5'-cagttgtaataagtaaaggataaacatgcgacctgttagacaaatcgcacagcttttcaattcatc-3') where the first 40 bases are homologous to the POL4 sequence just inside of the start and stop sites, respectively, of the POL4 coding region and the remaining bases are homologous to the 5' 16 bases (CHS6) and the 3' 20 bases (CHS5) of URA3 in the plasmid p426GPD (MUMBERG et al. 1995) used as template. Deletion of POL4 was identified by colony PCR using the primers CHS1 (5'-cactattcttctatctcc-3') and CHS2 (5'-ctttcctccttccccatcc-3') located 150 bp upstream of the start and stop codons in the Pol4 coding region. Briefly, cells were lysed by five cycles of 1 min freeze in dry ice/ethanol bath, 1 min thaw at 37° H2O. Fifty microliters of PCR reaction mix containing 10x PCR buffer (200 mM Tris-HCl pH 8.0, 250 mM KCl, 15 mM MgCl2, 0.5% Tween 20), 1.5 mM MgCl2, 0.2 mM dNTPs, 1.25 units of Taq (Promega, Madison, WI), 8 nM primer, and 1 ng template was added to the lysed cells and amplification conditions were as follows: 5 min at 95° followed by 40 cycles of 30 sec at 95°, 30 sec at 50°, 2 min at 72°, and a final extension of 10 min at 72°. The resulting products were resolved by electrophoresis on a 1% agarose gel (American Bioanalytical), an example of which is shown in Figure 1. The yku70::TRP strain was constructed as described above using primers CHS110 (5'-agctatgatttgttaagtgactctaagcctgattttaaaacgggaatatttggtgactattgagcacg-3') and CHS111 (5'-ctaccaaatattgtatgtaacgttatagatatgaaggatttcaatcgtctctccttacgcatctgtgc-3') where the first 40 bp are homologous to the YKU70 sequence just inside of the start and stop sites, respectively, of the YKU70 coding region, and the remaining base pairs are homologous to the 5' 18 bases and the 3' 18 bases of TRP1 in the plasmid pAS2.1 (CLONTECH, Palo Alto, CA), used as template in the PCR reaction. yku70::TRP1 colonies were detected, as described above, using primers CHS114 (5'-ttgttaagtgactctaagc-3') and CHS115 (5'-taacgttatagatatgaagg-3'), located 40 bases upstream and downstream of the YKU70 coding region. Wild-type YKU70 (1708 bases) and yku70::TRP1 (774 bases) PCR products were differentiated by gel electrophoresis as previously described.

    Transformations to create gene deletions or introduce plasmids were carried out by the lithium acetate procedure (ITO et al. 1983).

    Measurement of growth rates:

    To measure growth rates of wild-type and deletion strains, overnight cultures were diluted to optical density 600 nM = 0.1 and incubated with shaking at 30°. The optical density at 600 nM was measured at regular intervals.

    Sensitivity to DNA-damaging agents:

    The sensitivity of various strains to DNA damaging agents was characterized by spotting 5 μl of 10-fold serial dilutions (starting at 2 x 107 cells/5 μl) onto plates containing DNA-damaging agents at the indicated concentrations. To assess cell killing by MMS, overnight cultures were diluted and grown to 1 x 107 cells/ml. Appropriate serial dilutions were plated on YPD plates containing 0–0.06% MMS and colonies were counted after 3 days of growth at 30°. The percentage of survival was determined by dividing the total number of colonies grown in the presence of MMS by the total number of colonies grown in the absence of DNA damage.

    Measurements of spontaneous and MMS-induced mutagenesis using the canavanine-resistance assay:

    Forward mutation frequencies were determined on the basis of a previously described protocol (HADJIMARCOU et al. 2001). Briefly, cells were grown in 5 ml YPADU to saturation. Cells were harvested by centrifugation, washed once, and resuspended in 1 ml sterile water. One hundred microliters of various dilutions were spread on SC-Arg to determine the viable cell count and on SC-Arg containing canavanine, with and without 0.02% MMS, to determine the number of mutants arising spontaneously and induced by MMS treatment. Mutation frequency was calculated by dividing the total number of canavanine-resistant colonies by the total number of colonies plated.

    Canr mutation spectra:

    DNA from independent Canr colonies was isolated and the CAN1 gene was amplified using colony PCR as described above with the primers CANF1 and CANR1 (HUANG et al. 2002). PCR products were purified using the QIAGEN (Chatsworth, CA) PCR purification kit and sequenced with primers CHS84 (5'-tggccgcaccaaatgc-3'), CHS85 (5'-ggtttgcagcttcaccagc-3'), and CHS86 (5'-cgtggaaatgtgatcaaaggt-3') located inside the CAN1 coding region. DNA sequencing was performed at the Keck Facility at Yale University School of Medicine.

    Measurements of spontaneous and MMS-induced mutagenesis using the lysBglI frameshift assay:

    The lys2Bgl reversion frequency was obtained and isolation of revertants was performed as previously described (GREENE and JINKS-ROBERTSON 1997). Briefly, 2-day-old colonies were isolated from YEPD plates and inoculated into 5 ml YPEGE liquid. Cells were grown for 2 days at 30° shaking. Cells were harvested by centrifugation, washed once with 1 ml dH2O, and resuspended in 1 ml dH2O. One-hundred-microliter aliquots of the appropriate dilutions were plated in the presence and absence of 0.02% MMS on YEPD to determine the number of viable cells and on SC-lys to select Lys+ revertants. Frameshift frequency was calculated by dividing the number of Lys+ cells by the total number of cells plated on YEPD alone. A 900-bp region containing the lys2Bgl reversion window was amplified from the Lys+ revertants using colony PCR as previously described (GREENE and JINKS-ROBERTSON 1997). The amplified DNA was purified using the QIAGEN PCR purification kit and DNA sequencing was performed by the Keck Facility at Yale University School of Medicine.

    RESULTS

    Cells deleted of DNA polymerase 4 grow normally:

    To determine if deletion of Pol4 in cells affected growth, pol4 cells were assayed for viability, as described in MATERIALS AND METHODS. The pol4 cells are viable and deletion of POL4 does not affect the growth rate of the haploid or diploid strains as shown in Figure 2. Thus, consistent with previous findings, Pol4 is not required for normal growth of cells (PRASAD et al. 1993; LEEM et al. 1994; BUDD and CAMPBELL 1995).

    Sensitivity of Pol4-deficient S. cerevisiae to DNA-damaging agents:

    Although Pol4 is not required for normal cellular growth, previous work regarding its cellular role is controversial. Some work shows an important role for Pol4 in the repair of damage induced by the SN2 alkylation agent MMS in diploid cells (LEEM et al. 1994), whereas other groups reported no sensitivity to DNA-damaging agents (PRASAD et al. 1993; BUDD and CAMPBELL 1995; MCINNIS et al. 2002). It is important to note that pol4 strains that showed no sensitivity to MMS were haploid cells.

    To identify potential DNA repair pathway(s) in which Pol4 is involved, we tested the sensitivity of pol4 diploid strains with the SK1 genetic background to a variety of DNA-damaging agents as shown in Figure 3. Of the agents tested, we found that only pol4/pol4 strains were sensitive to MMS in this qualitative assay. We confirmed this to be the case in quantitative survival assays, an example of which is shown in Figure 4. Interestingly, MMS sensitivity is observed only in pol4 diploid cells with an SK1 strain background as compared to the BR19198b strain background, as shown in Figure 5.

    Pol4 may function in an end-joining pathway:

    Previous work with Pol4 indicated that Pol4 played a role in nonhomologous end joining of plasmid substrates particularly when end processing was required (WILSON and LIEBER 1999) and more recent work has identified two Pol4-interacting factors, Dnl4/Lif1 and Rad27. These proteins stimulate each other's function and operate in joining DNA substrates containing 5'-ends that require processing prior to joining (TSENG and TOMKINSON 2002, 2004). To determine if Pol4 functions in NHEJ, we analyzed the MMS sensitivity of pol4 cells in combination with yku70. YKu70 is a DNA end-binding protein involved in protecting ends from degradation during NHEJ and yku70 mutant strains have a dramatic decrease in end joining in the absence of homologous recombination (MILNE et al. 1996; SIEDE et al. 1996; CHEN et al. 2001; TOMITA et al. 2003; reviewed in DUDASOVA et al. 2004). In the strains that we examined, yku70 cells were not sensitive to MMS-induced DNA damage as shown in Figure 4. Deletion of YKU70 in pol4 cells rescued the MMS sensitivity of the pol4 strain (Figure 4). This suggests that YKU70 may be upstream of Pol4 in the repair of DNA damage and that elimination of KU70 results in repair by another pathway, perhaps homologous recombination.

    Pol4 is involved in maintaining genomic integrity during repair of DNA damage:

    To determine if Pol4 is important for accurate DNA repair, we characterized the frequency and types of mutations that arise in the absence of Pol4 using the canavanine assay in the SK1 strain background. POL4 was deleted in haploid strains JSY177 and JSY178 to create JSY199 and JSY200. The CanR spontaneous mutation frequency is similar for wild-type and pol4 cells in each of three different experiments, as shown in Table 2. Surprisingly, deletion of Pol4 results in a 6- to 14-fold increase in the MMS-induced CanR mutation frequency, as shown in Table 2. We sequenced the CanR mutants induced by MMS in experiment 2 (Table 2) to determine the types of mutations that arose in wild-type and pol4 cells. Analysis of the CanR mutants revealed that pol4 mutants have a mutation spectrum that is different from that of wild-type cells as shown in Figure 6. In fact, comparison of the wild-type and pol4 MMS-induced spectra by the Adams and Skopek algorithm suggests that they are significantly different (P < 0.001) (ADAMS and SKOPEK 1987). However, caution should be taken with this interpretation because large numbers mutants (>100) are not present in each of the spectra. The types of mutations induced by MMS are shown in Table 3. In the pol4 strain, the frequencies of each type of base substitution are increased over that of wild type. Most notably, we observed a 93-fold increase in AT-to-TA transversions in the Pol4-deficient cells. Analysis of the classes of mutations by the Adams and Skopek algorithm suggests that the only significant difference in mutational classes is AT to TA with a P-value of 0.011. We may also observe an increase in the frequency of TA-to-CG transitions and duplications in the pol4 cells. However, the numbers of observed mutations in these categories are small, so the significance of the calculated differences is not known. The duplications that we observe appear to be microhomology mediated, as suggested by the homologous sequence present upstream and downstream of both the duplications and deletions as shown in Figure 6.

    Pol4 is not a frameshift mutator:

    To determine if deletion of POL4 alters the frequency or types of frameshift mutations observed in wild-type cells, we compared frameshift mutagenesis of wild-type and pol4 strains using the lys2Bgl assay (GREENE and JINKS-ROBERTSON 1997). POL4 was deleted with URA3 in SJR335 and SJR357 to create JSY203 and JSY204. Frameshift mutations were measured as the number of viable Lys+ colonies. We found the spontaneous frameshift frequencies for haploid cells to be quite similar for wild-type and the pol4 strain in the presence and absence of 0.02% MMS in both haploid and diploid cells, as shown in Table 4. This suggests that deletion of Pol4 does not alter frameshift frequencies in vivo. Sequencing of the Lys+ colonies showed that the spectrum of frameshifts was similar in the wild-type and pol4 strains (data not shown).

    DISCUSSION

    The goal of our studies is to understand the role of S. cerevisiae DNA polymerase 4 in vivo. We show that cells that are deficient in Pol4 are sensitive to MMS in the diploid SK1 strain background. We also demonstrate that the MMS sensitivity of pol4 cells can be rescued by deletion of YKu70, a protein that is required for the initial steps of nonhomologous end joining. These results indicate that Pol4 may function in an end-joining pathway, downstream of Ku70, which is consistent with what has been observed in vitro (TSENG and TOMKINSON 2002, 2004). We also show that deletion of POL4 results in an increase in the MMS-induced mutation frequency. In the absence of Pol4, we observe a significant increase in AT-to-TA transversions. These results indicate that Pol4 is critical for accurate repair of DNA damage and, most likely, accurate repair of A adducts.

    Pol4 exhibits a phenotype in cells with the SK1 strain background:

    Before we initiated this project, a review of the literature suggested that only cells with an SK1 strain background exhibited an MMS-sensitive phenotype in the absence of Pol4. Our studies confirm these observations. The MMS sensitivity of SK1 diploid pol4 cells may reflect documented, but poorly understood, genetic differences between the SK1 strain background and other wild-type laboratory strains such as S288c, for which sequence information is available (PRIMIG et al. 2000). PRIMIG et al. (2000) reported 39 gene deletions and 2025 polymorphisms in SK1 compared to S288c and WILLIAMS (2002) reported 39 large deletions and 2025 differentially expressed transcripts in SK1 relative to S288c. Although analysis of the genes missing in SK1 cells does not reveal any proteins whose absence would clearly explain the observed MMS sensitivity of pol4/pol4 cells, perhaps some of the best candidates are genes encoding proteins involved in transport. For example, both YDR036c and YGL051w are deleted in SK1 cells and are involved in endocytosis and vesicle formation, respectively. Perhaps in their absence intracellular MMS accumulates. Additionally, absence of the transporter encoded by YDR038c could result in increased amounts of intracellular MMS in diploid cells of an SK1 background, leading to increased cellular damage.

    The Pol4-specific DNA repair pathway is important for survival of diploid cells:

    Deletion of Pol4 results in MMS sensitivity in diploid, but not haploid, cells. The MMS-sensitive phenotype can be rescued by deletion of YKU70. YKu70 is involved in the protection or alignment of ends when DNA breaks are repaired in an end-joining pathway. Our data are consistent with the interpretation that Pol4 functions downstream of Yku70 in an end-joining pathway in S. cerevisiae. We suggest that chronic exposure to MMS results in strand breaks. Once the ends of these breaks are aligned, Pol4 is likely to participate in gap filling. This suggestion is consistent with the finding that deletion of Pol4 results in a large deficiency in the joining of the ends of plasmids that contain breaks predicted to have 5' or 3' overhangs once they are aligned using microhomology (WILSON and LIEBER 1999). In our strains, elimination of KU70 most likely results in DNA damage being repaired by a KU- and Pol4-independent repair pathway. An alternative explanation is that because pol4 and ku70 are not epistatic, they are not members of the same DNA repair pathway. We do not favor this explanation because other studies have indicated a role for Pol4 in end joining (TSENG and TOMKINSON 2002, 2004).

    It is not entirely clear why diploid, but not haploid, cells require Pol4 to survive the effects of MMS. It is known that Nej1, which is important for localizing Lif1 to the nucleus, is regulated by the MATa/MAT repressor (VALENCIA et al. 2001) and that Lif1 interacts with Dnl4 (HERRMANN et al. 1998). Dnl4 and Lif 1 are homologs of mammalian DNA ligase IV/XRCC4 and function in ligation of DNA ends. Interaction of Lif1 with Dnl4 results in its stabilization. This results in the stabilization of Dnl4, targeting it to DSBs, and stimulating Dnl4 activity (TEO and JACKSON 2000). Control of Nej1 expression by the MATa/MAT repressor limits Nej1 expression to haploid cells. Therefore, it is thought that Dnl4 and Lif1 participate in NHEJ activities in haploid cells. If Pol4 functions in Ku-dependent NHEJ in diploid cells, our data suggest that Pol4 may function in a Nej1/Lif1-independent end-joining pathway. In SK1 diploid cells, Pol4 may target Dnl4 to sites of DNA damage independently of Lif1 and Nej1 or Pol4 may function with another ligase such as Cdc9 or a yet-unidentified ligase (WU et al. 1999; WANG et al. 2004).

    Pol4 is important for accurate DNA repair in haploid cells:

    To determine if Pol4 is important for genomic stability, we characterized spontaneous mutagenesis and the types of mutations that arose in the presence of a low dose of MMS. We found that spontaneous mutation frequencies did not vary between wild-type and pol4 cells but we observed a 6- to 14-fold increase in the MMS-induced mutation frequency of pol4 cells compared with wild-type cells. This suggests that Pol4 is important for accurate repair of DNA damage in haploid cells, even though it is not critical for their survival. Sequencing of the canavanine gene from CANR colonies that arose after exposure to MMS showed that the mutation spectrum of pol4 cells is significantly different from that of wild type. Analysis of the mutation spectra reveals an increase predominantly in the frequency of AT-to-TA base substitutions. Because MMS is an SN2 alkylating agent that forms 7 methylguanine and 3 methyladenine (3MeA) adducts (HOFFMANN 1980; SINGER and BRENT 1981), our data suggest that Pol4 is especially important for the accurate repair of 3MeA.

    The MMS-induced mutation spectrum suggests that 3MeA DNA adducts give rise to the AT-to-TA base substitution mutations that we observe. This lesion is known to interfere with DNA synthesis and to be extremely detrimental to cellular survival (RACINE et al. 1993; ENGELWARD et al. 1998; MORALES and KOOL 2000). Pol4 may function directly at the replication fork, in place of the replicative polymerases when the fork is blocked by a 3MeA adduct, although no direct evidence exists that supports this idea. Because Pol4 is not required for haploid cells to survive in the presence of MMS, our results suggest that another polymerase, perhaps a member of the Y family of translesion polymerases, is able to function in place of Pol4 at the fork, but that this results in the induction of mutations. Whether Pol4 is able to accurately bypass a 3MeA adduct, or whether it functions in repair synthesis at the replication fork, remains to be determined. An alternative explanation is that Pol4 functions in the gap-filling step of base excision repair (BER), because methylated base adducts are known to be repaired predominantly by BER (FREIDBERG et al. 1995). If this were the case, a polymerase other than Pol4, perhaps one that is error prone, would be predicted to function in its place, inserting T opposite template T once the methylated A has been excised.

    In the MMS-induced mutation spectra we also observed a possible increase in large duplications, which appears to be homology mediated. MA et al. (2003) identified a Ku independent end-joining pathway termed microhomology-mediated end joining (MMEJ), which is dependent upon the Mre11/Rad50/Xrs2 complex. In the absence of Pol4, homology-mediated duplications may occur through MMEJ. The duplications that we observe may result from strand displacement synthesis and lack of excision of the 5' flap. Interestingly, deletion of POL4 did not result in a change in the frequency of single base frameshift mutations. This indicates that Pol4 does not protect cells against these types of mutations or that redundant pathways may function in the absence of Pol4.

    In summary, we have shown that Pol4 plays an important role in the processing of MMS-induced DNA damage. In SK1 diploid cells the polymerase activity of Pol4 is important for repair of MMS-induced DNA damage. The diploid-specific requirement of Pol4 for survival may be attributed to a Nej1/Lif1 independent end-joining mechanism or an ability of Pol4 to interact with Cdc9 or another DNA ligase during end joining. Finally, we identified a critical role for Pol4 in accurate DNA repair. Treatment of pol4 cells with a low dose of MMS leads to an increase in base substitutions, namely AT-to-TA transversions and homology-mediated duplications. These findings suggest a role for Pol4 in accurate repair of DNA damage.

    ACKNOWLEDGEMENTS

    We thank Susan Baserga, Charles Radding, Michael Snyder, and Shirleen Roeder for their helpful advice. This work was supported by ES10995 from the National Institute of Environmental Health Sciences.

    LITERATURE CITED

    ADAMS, W., and T. SKOPEK, 1987 Statistical test for the comparison of samples from mutational spectra. J. Mol. Biol. 194: 391–396.

    ARAVIND, L., and E. V. KOONIN, 1999 DNA-binding proteins and evolution of transcription regulation in the archaea. Nucleic Acids Res. 27: 4658–4670.

    BORK, P., C. OUZOUNIS, C. SANDER, M. SCHARF, R. SCHNEIDER et al., 1992 Comprehensive sequence analysis of the 182 predicted open reading frames of yeast chromosome III. Protein Sci. 1: 1677–1690.

    BUDD, M. E., and J. L. CAMPBELL, 1995 Purification and enzymatic and functional characterization of DNA polymerase beta-like enzyme, POL4, expressed during yeast meiosis. Methods Enzymol. 262: 108–130.

    GUTHRIE, C., and G. R. FINK, 1991 Methods in Enzymology. Academic Press, San Diego.

    CHEN, L., K. TRUJILLO, W. RAMOS, P. SUNG and A. E. TOMKINSON, 2001 Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol. Cell 8: 1105–1115.

    DUDASOVA, Z., A. DUDAS and M. CHOVANEC, 2004 Non-homologous end-joining factors of Saccharomyes cerevisiae. FEMS Microbiol. Rev. 28: 581–601.

    ENGELWARD, B. P., J. M. ALLAN, A. J. DRESLIN, J. D. KELLY, M. M. WU et al., 1998 A chemical and genetic approach together define the biological consequences of 3-methyladenine lesions in the mammalian genome. J. Biol. Chem. 273: 5412–5418.

    FREIDBERG, E. C., G. C. WALKER and W. SIEDE, 1995 DNA Repair and Mutagenesis. American Society for Microbiology, Washington, DC.

    GREENE, C. N., and S. JINKS-ROBERTSON, 1997 Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins. Mol. Cell. Biol. 17: 2844–2850.

    HADJIMARCOU, M. I., R. J. KOKOSKA, T. D. PETES and L. J. REHA-KRANTZ, 2001 Identification of a mutant DNA polymerase in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations. Genetics 158: 177–186.

    HERRMANN, G., T. LINDAHL and P. SCHAR, 1998 Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4. EMBO J. 17: 4188–4198.

    HOFFMANN, G. R., 1980 Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds. Mutat. Res. 75: 63–129.

    HUANG, M. E., A. G. RIO, M. D. GALIBERT and F. GALIBERT, 2002 Pol32, a subunit of Saccharomyces cerevisiae DNA polymerase , suppresses genomic deletions and is involved in the mutagenic bypass pathway. Genetics 160: 1409–1422.

    ITO, H., Y. FUKUDA, K. MURATA and A. KIMURA, 1983 Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163–168.

    LEEM, S. H., P. A. ROPP and A. SUGINO, 1994 The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair. Nucleic Acids Res. 22: 3011–3017.

    MA, J. L., E. M. KIM, J. E. HABER and S. E. LEE, 2003 Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair breaks lacking overlapping end sequences. Mol. Cell. Biol. 23: 8820–8828.

    MCINNIS, M., G. O'NEILL, K. FOSSUM and M. S. REAGAN, 2002 Epistatic analysis of the roles of the RAD27 and POL4 gene products in DNA base excision repair in S. cerevisiae. DNA Rep. 1: 311–315.

    MILNE, G. T., S. JIN, K. B. SHANNON and D. T. WEAVER, 1996 Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 4189–4198.

    MORALES, J. C., and E. T. KOOL, 2000 Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases. Biochemistry 39: 12979–12988.

    MUMBERG, D., R. MULLER and M. FUNK, 1995 Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156: 119–122.

    PRASAD, R., S. G. WIDEN, R. K. SINGHAL, J. WATKINS, L. PRAKASH et al., 1993 Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme. Nucleic Acids Res. 21: 5301–5307.

    PRIMIG, M., R. M. WILLIAMS, E. A. WINZELER, G. G. TEVZADZE, A. R. CONWAY et al., 2000 The core meiotic transcriptome in budding yeasts. Nat. Genet. 26: 415–423.

    RACINE, J. F., Y. ZHU and M. D. MAMET-BRATLEY, 1993 Mechanism of toxicity of 3-methyladenine for bacteriophage T7. Mutat. Res. 294: 285–298.

    ROCKMILL, B., and G. S. ROEDER, 1990 Meiosis in asynaptic yeast. Genetics 126: 563–574.

    SHIMIZU, K., C. SANTOCANALE, P. A. ROPP, M. P. LONGHESE, P. PLEVANI et al., 1993 Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta. J. Biol. Chem. 268: 27148–27153.

    SIEDE, W., A. A. FRIEDL, I. DIANOVA, F. ECKARDTSCHUPP and E. C. FRIEDBERG, 1996 The Saccharomyces cerevisiae Ku auto-antigen homologue affects radiosensitivity only in the absence of homologous rcombination. Genetics 142: 91–102.

    SINGER, B., and T. P. BRENT, 1981 Human lymphoblasts contain DNA glycosylase activity excising N-3 and N-7 methyl and ethyl purines but not O6-alkylguanines or 1-alkyladenines. Proc. Natl. Acad. Sci. USA 78: 856–860.

    TEO, S. H., and S. P. JACKSON, 2000 Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks. Curr. Biol. 10: 165–168.

    TOMITA, K., A. MATSUURA, T. CASPARI, A. M. CARR, Y. AKAMATSU et al., 2003 Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double strand breaks but not telomeres. Mol. Cell. Biol. 23: 5186–5197.

    TSENG, H. M., and A. TOMKINSON, 2002 A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining. J. Biol. Chem. 277: 45630–45637.

    TSENG, H. M., and A. E. TOMKINSON, 2004 Processing and joining of DNA ends coordinated by interactions among Dnl4/Lif1, Pol4, and FEN-1. J. Biol. Chem. 279: 47580–47588.

    VALENCIA, M., M. BENTELE, M. B. VAZE, G. HERRMANN, E. KRAUS et al., 2001 NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae. Nature 414: 666–669.

    WANG, X., G. IRA, J. A. TERCERO, A. M. HOLMES, J. F. X. DIFFLEY et al., 2004 Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae. Mol. Cell. Biol. 24: 6891–6899.

    WILLIAMS, R. M., 2002 The yeast life cycle and DNA array technology. J. Ind. Microbiol. Biotechnol. 28: 186–191.

    WILSON, T. E., and M. R. LIEBER, 1999 Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway. J. Biol. Chem. 274: 23599–23609.

    WU, X., E. BRAITHWAITE and Z. WANG, 1999 DNA ligation during excision repair in yeast cell-free extracts is specifically catalyzed by the CDC9 gene product. Biochemistry 38: 2628–2635.

    Departments of Therapeutic Radiology and Genetics, Yale University, New Haven, Connecticut 06510(Catherine H. Sterling and)