A DNA Polymerase ε Mutant That Specifically Causes 11 Frameshift Mutations Within Homonucleotide Runs in Yeast

The DNA polymerases d and ε are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 39 → 59 exonuclease proofreading activity. Many errors arising during replication are corrected by these exonuclease activities. We have investigated the contributions of regions of Polε other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polε mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for 11 frameshifts while there was almost no effect on 21 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading. In combination with a deletion of the mismatch repair gene MSH2, the 11 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the 21 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes 11 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs). This is the first report of a 11-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polε. ERROR avoidance and correction are essential for the conserved amino acid motifs ExoI, ExoII, and ExoIII found at the amino-terminal end of B family polymerreducing a species’ mutational load. DNA polymerases are intrinsically accurate during replicative syntheases (Morrison et al. 1991; Simon et al. 1991; see Figure 1). sis due to both base selectivity and opportunities to correct errors through proofreading (Kunkel 1992). In Proofreading by either Pold or Polε is ineffective at correcting frameshift errors in long homonucleotide addition, mismatch repair (MMR) systems provide for postreplicational monitoring and correction of errors. runs during in vitro or in vivo replication (Kroutil et al. 1996; Tran et al. 1997). Such runs are especially In all eukaryotes examined nuclear DNA polymerases Polε and -d possess 39 → 59 exonuclease activity (Morprone to frameshift mutations, which are generally acknowledged to arise by replication slippage (reviewed in rison et al. 1991). Genetic studies in the yeast SaccharoGordenin and Resnick 1998). Two models for slippage myces cerevisiae have demonstrated that the 39 → 59 exohave been proposed. One model proposes that slippage nuclease activities of these polymerases are responsible occurs through disassociation of the polymerase from for proofreading of newly replicated DNA (Morrison the template and incorrect reannealing of the template et al. 1991; Simon et al. 1991; Morrison and Sugino and nascent strands (Streisinger et al. 1966; Kunkel 1994) and that proofreading mutants are frameshift and and Soni 1988). Recently an alternative model for slipbase substitution mutators (Morrison et al. 1991; Simon page leading to insertion frameshifts has been proet al. 1991). Proofreading by other replicative polymerposed. In this model frameshifts occur during reannealases can decrease DNA replication error rates up to two ing of the DNA strands after partitioning of the nascent orders of magnitude in vitro (Kunkel 1992) and in vivo strand between the polymerization and proofreading (Schaaper 1993). Proofreading activity is provided by domains (Fujii et al. 1999). The in vitro correction of mispaired bases by proofreading is limited to approximately five nucleotides behind the 39 terminus of the Corresponding author: Michael A. Resnick, National Institute of Enviprimer (Lam et al. 1999). Genetic studies have revealed ronmental Health Sciences (NIEHS), Mail Drop D3-01, 111 T.W. Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709. similar limitations on proofreading in vivo (Tran et al. E-mail: [email protected] 1997). 1 Present address: Department of Chemistry and Biochemistry, 601 The 59 → 39 exonuclease encoded by EXO1 is involved University Dr., Southwest Texas State University, San Marcos, TX in recombination, resistance to UV damage, and MMR 78666. 2 Present address: LifeSensors Inc., Malvern, PA 19355. (Fiorentini et al. 1997; Tishkoff et al. 1997a; Johnson Genetics 155: 1623–1632 (August 2000) 1624 J. M. Kirchner, H. Tran and M. A. Resnick et al. 1998; Qiu et al. 1998; Tran et al. 1999b) and have taken a similar in vivo approach in the generation complements many defects in rad27 strains (Tishkoff of DNA Polε mutations. In addition to functioning in et al. 1997b; Parenteau and Wellinger 1999). Rad27, cellular DNA replication and proofreading, Polε has the yeast homologue of human flap endonuclease been implicated in various types of DNA repair includ(FEN1), is a 59 → 39 exo/endonuclease responsible for ing nucleotide excision repair (NER), base excision rethe maturation of Okazaki fragments during lagging pair (BER), and recombination (Budd and Campbell strand DNA synthesis and removal of 59 flaps (reviewed 1997; Burgers 1998). We have developed a novel mutain Lieber 1997). Rad27 has also been proposed to function detection system, based on interactions with exo1 tion in MMR (Johnson et al. 1995). However, its role mutants, to identify functions of Polε other than proofin MMR has been questioned by the finding that in reading that influence genome stability. We have identiforward mutation assays the spectra of Drad27 and fied a unique DNA polymerase mutator with defects Dmsh2 mutants are very different (Tishkoff et al. that are not due to a change in proofreading capacity or 1997b). The absence of RAD27 can result in increased the ability to function in MMR. Unlike other mutators, it mutation rates including expansion of repeat sequences specifically causes 11 frameshift mutations in homonuand large duplications (Tishkoff et al. 1997b; Freudencleotide runs. reich et al. 1998; Kokoska et al. 1998; Maurer et al. 1998). Furthermore, a structure with mispaired nucleotides in close association with a 59 flap is processed by MATERIALS AND METHODS Fen to remove both the flap and the mispaired nucleoGeneral genetic and molecular methods: Standard yeast tides (Rumbaugh et al. 1999). Mutants lacking both media and yeast extract-peptone-dextrose (YPD) media with Exo1 and Rad27 are inviable (Tishkoff et al. 1997b; G418 have been described previously (Rose et al. 1990). Yeast Gary et al. 1999). Overexpression of Exo1 in a Drad27 cells were grown at 308 unless otherwise stated. Yeast transformutant complements the temperature sensitivity and mations were performed by the method of Gietz and Schiestl (1991). Preparation of bacterial growth media and partially complements the mutator phenotype of the molecular methods have been described previously (Tran et mutants (Tishkoff et al. 1997b; Parenteau and Welal. 1995, 1996). linger 1999) suggesting that Exo1 may also be able to Strains and plasmids: A series of isogenic strains was confunction directly in processing of flaps found in lagging structed from CG379 (Mata ade5-1 his7-2 leu2-3,112 trp1-289 strand replication intermediates. ura3-52) containing deletions of various DNA metabolism genes. Insertion of the InsE element with a homonucleotide Error avoidance in long homonucleotide runs is acrun containing varying lengths of A within LYS2 has been complished predominantly by MMR (Kolodner 1996; described (Tran et al. 1999b). The plasmid used to integrate Tran et al. 1997). The Escherichia coli methyl-directed mutations within POL2, p173, was constructed by subcloning mismatch repair system has provided a model for the the BamHI-BspEI fragment of POL2 from YCpPol2 (Morrison understanding of MMR in eukaryotes. The basic steps et al. 1990) into the BamHI-AvaI sites of pFL34*. pFL34* is of these MMR systems are similar, namely (1) mismatch identical to pFL34 (Bonneaud et al. 1991) except that the URA3 marker is in the opposite orientation after Bgl II digesrecognition, (2) incision of the newly replicated DNA tion (K. Lobachev, personal communication). Strains constrand, (3) nuclease-mediated deletion of the mismatch, taining pol2-C1089Y were constructed using a site-directed muand (4) gap filling and ligation. Similar to the E. coli tant of p173. Site-directed mutants were made using the system, in vitro results with human extracts have shown QuickChange mutagenesis kit from Stratagene (La Jolla, CA) that mismatch excision can occur from either side of a according to the manufacturer’s instructions. Primers to make the Cys to Tyr change and to add an RsaI restriction site were mismatch. In yeast the combination of a 39 → 59 Pold 59-GGTAAAAGATAAAGGTCTACAGTACAAATATATTATT or Polε exonuclease deficiency with a defect in either AGTCAAAACC and 59-GGTTTTGAACTAATAATATATTTG the EXO1 or the RAD27/FEN1 59 → 39 exonucleases TACTGTAGACCTTTATCTTTTACC. caused a synergistic increase in mutations in long homoMutagenesis of plasmid DNA: One tube containing plasmid nucleotide runs (Gary et al. 1999; Tran et al. 1999b). No p173 (pFL34* 1 POL2) was treated with 1 m hydroxylamine synergy was found between mutations in other mismatch in 50 mm pyrophosphate, 100 mm NaCl, pH 7.0, for 1 hr at 708. After stopping the reaction on ice, the DNA (3 mg) was repair genes (deletion of MSH2, MSH3, MSH6, or dialyzed against TE and transformed into DH5a cells. DNA PMS1) and deficiencies in proofreading (Pold or Polε) was isolated using a QIAGEN (Chatsworth, CA) column as for mutations occurring in long homonucleotide runs. described by the manufacturer. Since cells were grown for On the basis of these results it was proposed that the multiple generations mutations at the same sites cannot be exonuclease activities of Exo1, Rad27, and Polε or Pold considered independent. participate in and substitute for each other at the exciScreen for A12 homonucleotide run mutators: The mutagenized plasmid DNA was digested with AgeI or PimAI and transsion step of MMR (Tran et al. 1999b). formed into cells, which were plated to uracil drop-out media. Relatively little is known about the regions of eukaryoAfter 3 days of growth at 308 colonies were replica plated to tic DNA polymerases that determine DNA replication uracil and to lysine drop-out media. After 3 days of growth, accuracy in vivo. One approach is to create mutations colonies that exhibited four or more papillae on the lysine within the polymerases and relate structural changes to drop-out medium were picked from the uracil drop-out plate and streaked for single colonies. Mutator colonies were grown specific mutation-generating characteristics in vitro. We 1625 Polε Insertional Frameshift Mutator on 5-FOA and those that had lost the URA3 gene and were still mutators were used for further study. Gene replacement and disruptions: We used p173-rsa, described above, for replacement of the wild-type POL2 gene with the C1089Y allele. The plasmid was digested with AgeI or PimAI and transformed into cells with selection on uracil dropout media. Transformants were screened for the presence of the site-directed change by a two-step procedure. After PCR amplification the fragment was digested with RsaI to determine if the change was present. Strains that had the mutation were grown on 5-FOA to select for loss of the URA3 gene. Other strains containing DNA repair/replication mutants were made from previously described strains (Tran et al. 1997, 1999b). Mapping and DNA sequence analysis of mutants: The locations of the new POL2 mutations were determined by mapping using gap repair with deleted versions of p173. After determining that the mutations were between the AspI and SpeI sites of plasmid p173, DNA sequence analysis was performed on Figure 1.—(A) Domain structure of the protein coded for genomic DNA from this area. Six DNA fragments were made by DNA Pol2 cDNA and location of the changed amino acid in by PCR amplification using primers pairs (numbered from the mutator allele. Regions of Polε including the exonuclease the ATG with 1 indicating 59 → 39 strand and 2 indicating domains, the polymerization domain, and the zinc finger dothe 39 → 59 strand; all 20 nucleotides): 1, pol211949 and main are shown relative to the sequenced mutation. Shown pol223300; 2, pol212948 and pol223800; 3, pol213220 and underneath is the region that was mutagenized in this study. pol224030; 4, pol213520 and pol224342; 5, pol213826 and (B) Multispecies alignment of the region containing amino pol225000; 6, pol214100 and pol225000. Sequencing of the acid C1089. The Cys residue at amino acid 1089 of S. cerevisiae PCR fragments was done using an ABI model 373A DNA is in a 17-amino acid block that is highly conserved. When sequencer. Sequencing of Lys revertants was done by PCR four or more species have an identical amino acid in a given amplification of a fragment of the LYS2 gene that included location this position is considered conserved and it is highthe InsE insertion that contained the homonucleotide run. lighted in this figure. The primers used to amplify and sequence this fragment and the his7-2 homonucleotide run have been described (Shcherbakova and Kunkel 1999; Tran et al. 1999b). Measurement of mutation rates: Mutation rates were deterScreening of .5000 Ura transformants yielded 3 mined by a fluctuation test using the method of the median with significantly higher Lys reversion rates. Their mu(Lea and Coulson 1949) on at least 12 independent cultures tator phenotype was complemented by the plasmid as described (Tran et al. 1999b). YCpPol2 containing the wild-type POL2 gene. Isolates 37a and 45f were characterized further (the third muRESULTS tant is under study). The role of the EXO1 defect in revealing the POL2 mutator phenotype was determined Isolation of Polε mutators: Proofreading is required by introducing EXO1 into cells on the 2m plasmid for accurate replication by Polε. Little is known about pRDK480. The relative mutation rates of both of the the contributions of other regions of this polymerase pol2 exo1 mutants compared to wild type were increased to replication fidelity. We investigated the ability of ran33-fold. Addition of the pRDK480 plasmid reduced the domly generated Polε mutations, external to the known relative rates to 2and 3-fold over wild type, respectively. proofreading domain, to increase the mutation rate in This established that the pol2 mutants isolated are at best the long A12 homonucleotide run of the lys2::InsE-A12 weak lys2::InsE-A12 mutators on their own and require a allele. Such runs are highly sensitive to subtle changes deficiency in the EXO1 gene for their strong mutator in proteins that can affect the appearance of mutations phenotype in this assay. (Tran et al. 1997, 1999a,b; Clark et al. 1999; DrotschIdentification of the alterations within Polε: The Polε mann et al. 1999). The lys2::InsE-A12 allele was used premutator alleles were mapped initially by gap repair. viously in the characterization of various DNA metabolic Mutations 37a and 45f were localized to the same 1 kb mutants for their ability to influence 11 frameshift muof DNA. DNA sequencing revealed that both clones had tations (Tran et al. 1999a,b). a G-to-A change at base 3266 (C1089Y) of the coding The Polε mutants were examined in a strain conregion, suggesting a common origin for this mutation taining a deletion of EXO1. Use of such a genetically in these strains (Figure 1A). Alignment of Polε homosensitized background (Perkins et al. 1999) can increase logues from S. cerevisiae, Schizosaccharomyces pombe, Mus the probability of finding mutants when screening large musculus, Caenorhabditis elegans, Emericella nidulans, Arabinumbers of random isolates. On the basis of the results dopsis thaliana, and Homo sapiens identified a common with other double mutants (Tran et al. 1999b), we precysteine residue in a 17-amino acid block of homology dicted that mutant identification in the Dexo1 strain (Figure 1B) that is between 60 and 100% identical in would be more efficient than in the wild-type strain these organisms designated as C-2 (Huang et al. because of anticipated synergistic interactions between Polε mutations and the deletion allele of EXO1. 1999a,b). The cysteine to tyrosine change at amino acid 1626 J. M. Kirchner, H. Tran and M. A. Resnick 1089 is located 83 amino acids downstream from the bined with a mutation in the MMR gene MSH2, possibly due to the already high mutation rate of the msh2 mulast of the polymerase domains (Figure 1A) in a region that has been proposed to be involved in subunit interactant in this assay. The effect of the pol2-C1089Y mutation does not appear to extend to recombination since there tions (Kesti et al. 1993). To demonstrate that the C1089Y substitution is the was no effect in a plasmid-based recombination assay (J. M. Kirchner and M. A. Resnick, unpublished obonly alteration in POLε required for the mutator phenotype, we made two site-directed mutant constructs of servations). The POL2-C1089Y protein causes 11 frameshift muplasmid p173. The first construct had only the change found in the mutator allele. The second construct also tations in long homonucleotide runs: As shown in Table 1, the combination of pol2-C1089Y with Dexo1 or Drad27 contained silent changes giving rise to an RsaI restriction site. Either plasmid was sufficient to yield the mutacaused a .25-fold increase in reversion of the his7-2 allele. The proofreading defect pol2-4 leads to similar tor phenotype observed in the original mutant (data not shown). In our subsequent analysis we utilized strains synergistic increases in 21 frameshifts within long homonucleotide runs (Tran et al. 1999b; Table 1). We containing the p173-Rsa construct; this allele is referred to as pol2-C1089Y. Plating efficiencies or growth rates therefore examined combinations of Dexo1 or Drad27 with pol2-C1089Y for their effects on reversion of lys2 at 308 or 378 were not altered by this allele alone or in combination with Dexo1 (data not shown). alleles containing A10 or A14 runs. Surprisingly, pol2C1089Y allele did not lead to increased mutation rates in Impact of the Polε mutator in combination with other mutations: The Polε defect was examined on its own these 21 frameshift mutation detection assays, whereas pol2-4 resulted in z34and 4-fold increases in mutation and in combination with mutations in other genes that impact on the maintenance of homonucleotide runs. rates, respectively, when combined with Dexo1 or Drad27 in the lys2::InsE-A14 reversion assay (Table 1). Isogenic strains that contained A10, A12, and A14 runs within the LYS2 gene or an A7 run in the HIS7 gene To determine if the mutations scored in the lys2::InsEA12 assay were actually 11 frameshifts within the homo(i.e., his7-2) were used. Revertants of the lys2 alleles can occur only in a 79-bp window (Tran et al. 1997); nucleotide run, we sequenced Lys revertants of the pol2-C1089Y Dexo1 strain. As expected, all revertants reversion of the his7-2 allele occurs in a 43-bp window (Shcherbakova and Kunkel 1999). Defects in MMR (12/12) of the pol2-C1089Y Dexo1 strain in the lys2::InsEA12 assay were due to 11 frameshifts. Similarly, all mutaresult in all (A10, A12, and A14) or nearly all (his7-2) reversions occurring in these runs (Tran et al. 1997, tions that occurred in the his7-2 homonucleotide run in the pol2-C1089Y Dexo1 strain were associated with 11 1999b; Shcherbakova and Kunkel 1999). Revertants associated with the A12 and A7 runs are due to 11 mutations (10/10 sequenced). Thus, when assayed for frameshift mutator activity in long homonucleotide frameshifts and A14 and A10 revertants arise by 21 frameshifts. As shown in Table 1 the pol2-C1089Y mutaruns, the pol2-C1089Y allele leads to a specific increase in 11 mutations. tion itself does not increase mutation rates in the lys2::InsE-A12, A7, A10, and A14 homonucleotide runs. In The pol2-C1089Y mutator is not due to a proofreading defect: The 39 → 59 proofreading exonuclease activity contrast, the weak mutator phenotype for forward mutation rate at the CAN1 locus is increased relative to that of Polε (as well as Pold) can greatly reduce the potential for mutations. To determine if the pol2-C1089Y allele in POL strains. The mutator effect of pol2-C1089Y at CAN1 is greater than that observed for the proofreading alters proofreading, even though the substituted amino acid is outside the known exonuclease domains, this mutant pol2-4 (Morrison and Sugino 1994; Tran et al. 1999b; Table 1). mutant was examined for several phenotypic characteristics common to proofreading mutants. In comparison When combined with other DNA replication/repair defects, the pol2-C1089Y mutation was similar to pol2-4 with the single mutants, the double mutant pol2-4 msh2 exhibits a synergistic increase in CAN1 forward mutation for frameshifts in the A12 run. Both the Exo1 and Rad27 59 → 39 exonucleases have been implicated in mismatch rates (Morrison and Sugino 1994; Tran et al. 1999b). In pol2-C1089Y msh2 strains the mutation rate in the repair (Johnson et al. 1995) although this is controversial (Tishkoff et al. 1997b). The pol2-4 rad27 and pol2-4 CAN1 forward mutation assay is not significantly different from the msh2 single mutant (Table 1). The combiexo1 double mutants exhibit synergistic increases in mutation rates for homonucleotide runs, as well as at the nations pol2-4 exo1 or pol2-4 rad27 also result in synergistic increases in CAN1 mutation rates; however, no CAN1 locus, relative to the single mutants (Tishkoff et al. 1997b; Tran et al. 1999b). Results for double mutants synergy is observed for the corresponding pol2-C1089Y double mutants (Table 1). with pol2-C1089Y are presented in Table 1. Double mutants containing pol2-C1089Y and either Dexo1 or Drad27 Inactivating the 39 → 59 exonuclease activity of Polε does not increase mutation rates in long homonucleoalso led to synergistic (15to 20-fold) increases in the mutation rates in the lys2::InsE-A12 homonucleotide run. tide runs (Kroutil et al. 1996; Tran et al. 1999b; Table 1). This suggests that Polε acts together with other There was no significant enhancement of the lys2::InsEA12 mutation rate when pol2-4 or pol2-C1089Y was comnucleases, such as Exo1 and Rad27, to reduce mutations 1627 Polε In setion al Fram sh ft M uator TABLE 1 Interaction of pol2-C1089Y with mutations affecting error avoidance: increases in mutation rates for reversion in homonucleotide runs (A10, A12, A14 of lys2::InsE; A7 of his7-2) and forward mutation in CAN1 Mutation rates (3 10) A12 (11) A7 (11) A14 (21) A10 (21) CAN1 Mut. Rel. Mut. Rel. Mut. Rel. Mut. Rel. Mut. Rel. Genotype rate rate rate rate rate rate rate rate rate rate Wild type 14 (12–21) 1 2.3 (1.6–3.2) 1 19 (15–44) 1 13.9 (7.9–21) 1 24 (6.1–27) 1 pol2–4 15 (8–19) 1 nd nd 40.4 (24–71) 2 25 (14–50) 2 30 (15–83) 1 pol2–C1089Y 31 (24–41) 2 3.7 (3.2–6.7) 2 50 (31–83) 3 13 (11–19) 1 99 (70–110) 4 Dexo1 28 (17–30) 2 9 (7.2–16) 4 500 (326–875) 26 89 (76–130) 6 160 (110–270) 7 1pol2-4 1.6K (1K–2.3K) 114 nd nd 17.2K (10K–24K) 905 nd nd 890 (620–1.7K) 37 1pol2-C1089Y 463 (360–550) 33 362 (180–450) 158 746 (570–930) 39 54.5 (38–75) 4 257 (170–390) 11 Drad27 390 (292–448) 28 38.6 (32–58) 17 950 (710–2K) 50 162 (140–240) 12 240 (160–410) 10 1pol2-4 3.0K (2K–5.2K) 214 nd nd 3.5K (2K–5.7K) 184 nd nd 3.6K (2850–5740) 150 1pol2-C1089Y 6.3K (3.7K–9K) 452 1.0K (630–1.6K) 435 667 (470–890) 35 156 (115–240) 12 556 (460–690) 23 Dmsh2 10.0K (6.5K–32K) 714 450 (380–750) 195 220K (150K–340K) 11000 35.7K (22K–48K) 2570 616 (423–925) 25 1pol2-4 28.5K (25K–53K) 2035 13.9K (9.7K–36K) 6035 435K (330K–770K) 23000 17.3K (16.6K–45K) 1245 11.4K (7.4K–32K) 475 1pol2-C1089Y 50.0K (16K–75K) 3570 3.9K (2.3K–10K) 1696 450K (260K–1250K) 24000 10.7K (4.9K–13K) 770 1.9K (690–2150) 79 a The mutation rate of the strain designated was calculated by the method of the median (Lea and Coulson 1949). b 11 indicates mutations that occur by 1 base insertion, 21 indicates mutations that occur by 1 base deletion. c Rel. rate indicates the relative mutation rate compared to the wild-type strain for the given homonucleotide run. d The numbers in parentheses are the 95% confidence intervals for the mutation rate data. e nd indicates that the rate was not determined. f Large numbers are given with K to represent 1000s. 1628 J. M. Kirchner, H. Tran and M. A. Resnick