Emergence of DNA Polymerase e Antimutators That Escape Error-Induced Extinction in Yeast

DNA polymerases (Pols) e and d perform the bulk of yeast leadingand lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol d proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol e proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2D, mlh1D, msh3D msh6D, or pms1D mlh3D) but not with partial MMR loss (msh3D, msh6D, pms1D, or mlh3D), indicating that high levels of unrepaired Pol e errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2D eex mutants encoded second-site changes in Pol e that reduced the pol2-4 mutator phenotype between 3and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol e and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leadingand lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol e gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity. ORGANISMS must accurately duplicate their genomes to avoid loss of long-term fitness. Consequently, cells employ high-fidelity DNA polymerases (Pols) equipped with proofreading exonucleases to replicate their DNA (reviewed in McCulloch and Kunkel 2008; Reha-Krantz 2010). Mismatch repair (MMR) further ensures the integrity of genetic information by targeting mismatches for excision from newly replicated DNA (reviewed in Kolodner and Marsischky 1999; Iyer et al. 2006; Hsieh and Yamane 2008). These polymerase error-correcting mechanisms, together with DNA damage repair (Friedberg et al. 2006), maintain the genome with less than one mutation per 109 nucleotides per cell division (Drake et al. 1998). Defects in proofreading or MMR result in mutator phenotypes characterized by increased rates of spontaneous mutation (Kolodner and Marsischky 1999; Iyer et al. 2006; Hsieh and Yamane 2008; McCulloch and Kunkel 2008; RehaKrantz 2010). Mutator phenotypes can be an important source of genetic diversity, which facilitates adaptation to environmental change. In bacterial and yeast populations, unstable environments favor mutator strains that readily acquire adaptive mutations (Chao and Cox 1983; Mao et al. 1997; Sniegowski et al. 1997; Giraud et al. 2001a; Notley-McRobb et al. 2002; Nilsson et al. 2004; Thompson et al. 2006; Desai et al. 2007). In mammals, mutator phenotypes are proposed to accelerate the process of somatic cell evolution during tumorigenesis (Loeb et al. 1974, 2008). Deep sequencing of spontaneous tumors provides evidence for a mutator phenotype (Fox et al. 2009; Loeb 2011), and genetic defects in MMR or Pol proofreading elevate cancer susceptibility (Wei et al. 2002; Peltomäki 2005; Preston et al. 2010). However, mutator phenotypes do not persist indefinitely. Loss of fitness accompanies sustained expression of a mutator phenotype in a variety of organisms, including viruses (Smith et al. 2005), bacteria (Funchain et al. 2000; Giraud et al. 2001a), yeast (Wloch Copyright © 2013 by the Genetics Society of America doi: 10.1534/genetics.112.146910 Manuscript received October 21, 2012; accepted for publication December 5, 2012 Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.112.146910/-/DC1. Corresponding author: Department of Pathology, Box 357705, University of Washington, 1959 NE Pacific St., Seattle, WA 98195-7705. E-mail: [email protected] Genetics, Vol. 193, 751–770 March 2013 751 et al. 2001; Zeyl and De Visser 2001; Herr et al. 2011a), worms (Estes et al. 2004), and mammals (Albertson et al. 2009). Thus, following adaptation, selection pressure favors restoration of low mutation rates, which can occur through the elimination of mutator alleles or the acquisition of mutator suppressors (i.e., antimutators). A limited number of antimutator variants in the DNA replication machinery have been described (reviewed in Herr et al. 2011b). In the budding yeast Saccharomyces cerevisiae, DNA polymerases epsilon (Pol e) and delta (Pol d) are thought to perform the bulk of leadingand lagging-strand DNA synthesis, respectively (Pursell et al. 2007; Kunkel and Burgers 2008; Nick McElhinny et al. 2008; Larrea et al. 2010; Pavlov and Shcherbakova 2010). Both polymerases are accurate and possess intrinsic proofreading exonucleases that edit mispaired primer termini during polymerization (Morrison et al. 1991; Simon et al. 1991; Shimizu et al. 2002; Shcherbakova et al. 2003; Fortune et al. 2005). Defects in Pol e or Pol d proofreading increase the spontaneous mutation rate in a manner consistent with major roles for these polymerases in leadingand lagging-strand synthesis (Morrison et al. 1991; Simon et al. 1991; Morrison and Sugino 1994; Shcherbakova et al. 1996; Tran et al. 1999; Karthikeyan et al. 2000; Greene and Jinks-Robertson 2001). Interestingly, the Pol d proofreading defect generates a mutator phenotype 5to 30-fold greater than that observed in Pol e proofreading-deficient strains (Morrison and Sugino 1994; Shcherbakova et al. 1996; Tran et al. 1999; Datta et al. 2000; Karthikeyan et al. 2000; Greene and Jinks-Robertson 2001; Pavlov et al. 2004), and the spectra of spontaneous mutations that arise in Pol e and Pol d proofreading-deficient strains differ, which may reflect distinct error specificities of the polymerases as well as strand-specific effects (Morrison and Sugino 1994; Karthikeyan et al. 2000; Pavlov et al. 2002, 2003; Shcherbakova et al. 2003; Fortune et al. 2005). Mouse cells with defects in Pol e or Pol d proofreading also exhibit increased mutation rates (Goldsby et al. 2002; Albertson et al. 2009), and, consistent with distinct roles in DNA replication, the types of tumors that develop in Pol e and Pol d proofreading-deficient mice differ markedly (Albertson et al. 2009). Thus, avoidance of errors during eukaryotic DNA replication depends on both Pol e and Pol d proofreading (McCulloch and Kunkel 2008; Preston et al. 2010). The extent to which Pol e and Pol d proofreading contribute to DNA replication fidelity is obscured by MMR. The eukaryotic MMR machinery consists of homologs of bacterial MutS and MutL proteins (reviewed in Iyer et al. 2006; Hsieh and Yamane 2008). MutS homolog 2 (Msh2) associates with Msh6 or Msh3 to form two different heterodimers with partially overlapping activities. Msh2-Msh6 recognizes and binds to base-base and small insertion/deletion mispairs, while Msh2-Msh3 recognizes small and larger insertion/ deletion mispairs and a subset of base-base mispairs (Harrington and Kolodner 2007). Once bound to mismatched DNA, the Msh proteins recruit heterodimers of MutL homologs (Mlh). Mlh1 is the common subunit for two complexes. Mlh1-Pms1 (Mlh1-Pms2 in mammals) functions with both Msh2-Msh6 and Msh2-Msh3, while Mlh1-Mlh3 works primarily with Msh2-Msh3. Pms1 and Mlh3 contain latent endonucleases that cleave the nascent DNA strand, providing entry points for removal of mismatches and error-free DNA resynthesis (Kadyrov et al. 2006, 2007; Nishant et al. 2008). Consistent with these biochemical properties, genetic studies in yeast reveal overlapping mutator phenotypes when individual MMR genes are deleted. Deletion of MSH2 eliminates both Msh6and Msh3-dependent repair, effectively abrogating MMR and conferring a strong base-substitution and frameshift mutator phenotype. Deletion of MSH6 or MSH3 alone only partially inactivates MMR. msh6D mutants are strong base-substitution but weak frameshift mutators, msh3D strains are weak frameshift and duplication/deletion mutators (with increases in some base substitutions), and msh3D msh6D double mutants recapitulate the strong mutator phenotype of msh2D (Reenan and Kolodner 1992; New et al. 1993; Johnson et al. 1996; Marsischky et al. 1996; Greene and Jinks-Robertson 1997; Sia et al. 1997; Flores-Rozas and Kolodner 1998; Tran et al. 1999; Harrington and Kolodner 2007). Similar to msh2D, deletion of MLH1 inactivates MMR, resulting in a strong base-substitution and frameshift mutator phenotype. pms1D mutants are also strong basesubstitution and frameshift mutators, while mlh3D strains are weak frameshift and duplication/deletion mutators (Williamson et al. 1985; Strand et al. 1993; Prolla et al. 1994; Greene and Jinks-Robertson 1997; Flores-Rozas and Kolodner 1998; Yang et al. 1999; Harfe et al. 2000; Harrington and Kolodner 2007). Yeast pms1D mlh3D double mutants have strong mutator phenotypes, similar to or stronger than pms1D and mlh1D single mutants (FloresRozas and Kolodner 1998). In mice, deletion of both Mlh3 and Pms2 (equivalent to yeast PMS1) is required to recapitulate the strong mutator and cancer phenotypes caused by deletion of Mlh1 alone (Chen et al. 2005). Collectively, these studies indicate that Msh2-Msh6 and Mlh1-Pms1 (Mlh1-Pms2 in mammals) are the primary complexes that function in eukaryotic MMR, while Msh2-Msh3 and Mlh1Mlh3 play important secondary roles. Elimination of MMR in Pol e or Pol d proofreadingdeficient cells results in a multiplicative increase in mutation rate in diploid yeast, suggesting that proofreading and MMR act in series to correct polymerase errors (Morrison et al. 1993; Morrison and Sugino 1994). In haploids, combined inactivation of Pol d proofreading (via the pol3-01 allele) and any one of several MMR components (msh6D, msh2D, or pms1D) is lethal, presumably due to unrestrained mutagenesis during replication (Morrison et al. 1993; Tran et al. 1999; Greene and Jinks-Robertson 2001). We recently used a collection of pol3 mutator alleles to define the maximal mutation rate compatible with haploid yeast viability (Herr et al. 2011a). Cell populations become inviable when mutation rates exceed 1023 inactivating mutations/gene/cell division. This “error-induced extinction” (EEX) phenotype is readily suppressed by antimutator mutations encoding amino-acid substitutions in the catalytic subunit of Pol d, 752 L. N. Williams, A. J. Herr, and B. D. Preston as well as suppressor mutations in undefined genes. Thus, variants that escape error-induced extinction (eex mutants) provide a means to probe mechanisms of adaptation to mutator phenotypes (Herr et al. 2011a,b). Whether Pol e errors are also sufficient to trigger errorinduced extinction remains unclear. Morrison and Sugino (1994) reported that the pol2-4 allele, which inactivates Pol e proofreading, was not synthetically lethal with pms1Δ in haploid yeast, but noted that the resulting colonies were heterogeneous in size and grew slowly. Interestingly, pol2-4 pms1D isolates exhibited varying mutation rates, suggesting that mutator suppressors may arise in these strong mutator strains. Tran et al. (1999) also described haploid strains with Pol e proofreading and MMR defects: pol2-4 msh2D and pol24 msh3D msh6D. Mutation rate increases relative to pol2-4, msh2D, andmsh3Dmsh6D strains were consistent with a multiplicative relationship between MMR and Pol e proofreading (Tran et al. 1999). However, Greene and Jinks-Robertson (2001) later reported that they could not obtain a viable pol2-4 msh2D strain using either gene disruption or plasmid-shuffling methods. Thus, it remains unresolved whether the magnitude of Pol e errors is sufficient for error-induced extinction. Here, we show that defective Pol e proofreading is lethal to haploid yeast in the absence of MMR, providing evidence that, when left unrepaired, leading-strand errors exceed a mutation threshold. Moreover, we show that spontaneous mutants escape this Pol e error-induced extinction and that eex alleles function as antimutators. We discuss possible mechanisms of escape and mutator suppression. Our studies corroborate the unstable nature of mutators and provide a tractable system to investigate adaptive antimutator mutations that influence leading-strand DNA replication fidelity. Materials and Methods Media and growth conditions Standard media and growth conditions were used in the propagation of yeast strains (Sherman 2002). Cells were grown nonselectively using YPD or synthetic complete (SC) media with 2% dextrose. Selective growth was on SC media containing 2% dextrose and lacking the appropriate amino acid(s). Preformulated SC amino acid supplement was purchased from Bufferad, and supplements lacking defined amino acids were made from individual components as described (Sherman 2002). URA3-deficient cells were selected with 5fluoroorotic acid (FOA, 1 mg/ml; Zymo Research) media (Boeke et al. 1984). can1mutants were selected on SC lacking arginine and supplemented with 60 mg/ml of canavanine. Unless otherwise specified, reagents were purchased from SigmaAldrich or Fisher Scientific. Plasmids and strain constructions POL2 plasmids: pRS416, a CEN6/ARS4/URA3 plasmid (Brachmann et al. 1998), was engineered to carry the wildtype POL2 gene under control of its native promoter. The genomic sequence of POL2 was amplified from BY4733 yeast using Expand Hi-Fidelity DNA polymerase (Roche) and the following primers and PCR conditions: Pol2-XhoI (59ACTCGGTACTCGAGGCGCTCTGCCCTAGTTGGAATG-39; XhoI site underlined) and PolED2 (59-GATATTCCGAGCTCG CAACTTCCGGAGTGGTCAC-39; SacI site underlined); 94 , 1 min; 29· (94 , 15 sec; 58 , 20 sec; 68 , 8 min); 68 , 16 min. The resulting 7.5-kb fragment and pRS416 were digested with SacI and XhoI, ligated together with T4 DNA ligase (Gibco BRL), and then introduced into Escherichia coli. Transformed clones were isolated, and a correct POL2-containing plasmid was confirmed by sequencing the entire insert. This vector (pRS416POL2-59YIF1) did not fully complement the growth deficiency of our pol2D mutants. pRS416POL2-59YIF1 contains the entire POL2 coding sequence as well as 592 bp of upstream sequence, including 371 bp of noncoding sequence containing the promoter and 221 bp of the 59 end of the YIF1 gene, transcribed in the opposite direction. We hypothesized that transcription of the truncated YIF1 gene may suppress POL2 expression in our vector. Thus, we eliminated the YIF1 sequences by replacing the XhoI-SalI 59 fragment of pRS416POL2-59YIF1 with DNA amplified from this plasmid using PCR primers Pol2-7386bpXhoIF (59-ATGACTCGAGGTATGGGCCTTTGGTTTTCGT-39) and Pol2-8161bpR (59-GTTACACGCAATAAAGAAGTATGG39). The PCR product and pRS416POL2-59YIF1 were digested with BamHI and SalI and ligated together, and E. coli were transformed with the ligation product. The entire POL2 gene from a transformant was again sequenced to verify its integrity, and this new vector (pRS416POL2) fully complemented the growth deficiency of pol2D yeast. We subcloned the functional POL2 fragment from pRS416POL2 into the XhoI and SalI sites of the related plasmid pRS415 (CEN6/ ARS4/LEU2) (Brachmann et al. 1998) to obtain pRS415POL2. pRS416POL2 and pRS415POL2 contain the full-length POL2 coding sequence plus 368 bp upstream of the POL2 start site (corresponding to nucleotides 147844 to 155125 of yeast chromosome XIV). The pol2-4 mutation and all eex mutations were introduced into pRS415POL2 using the primers listed in supporting information, Table S1 (see also References for Supporting Tables) the QuikChange protocol (Wang and Malcolm 1999); Phusion polymerase (New England Biolabs); and the following PCR conditions: 95 , 1 min; 16· (95 , 40 sec; 53 , 1 min; 68 , 7 min). The entire pol2 gene was sequenced in each case to verify the presence of desired mutations and the absence of other mutations. All Pol d-expressing plasmids are previously described (Herr et al. 2011a). pRS vectors (Brachmann et al. 1998) were used as templates for chromosomal gene disruptions (see below). pUG6 served as a template for kanMX (Guldener et al. 1996) and pFv199 as a template for natMX (Stulemeijer et al. 2011). Strains: BY4733 and Y7092 haploid yeast strains were engineered to carry alleles of DNA polymerase and MMR genes (Table S2). BY4733 (MATa leu2D0 ura3D0 met15D0 trp1D63 his3D200), a S288C descendant (Brachmann et al. Antimutator Variants of DNA Polymerase e 753 1998), was obtained by sporulating a BY4733 · BY4734 diploid (kindly provided by Tim Formosa, University of Utah). All engineered BY4733 strains (Table S2) originated from the same spore. Y7092 (MATa can1D::STE2pr_his5 lyp1D ura3D0 leu2D0 his3D1 met15D0), also a S288C descendant, is a BY4742 derivative (Brachmann et al. 1998) modified by Boone and colleagues to use in synthetic genetic array analyses (Tong and Boone 2007). Chromosomal gene disruptions were made using PCR products generated with Phusion polymerase (New England Biolabs) and the primers, templates, and PCR conditions indicated in Table S3. Yeast were transformed with the resulting PCR products using lithium acetate transformation (Gietz and Woods 2002). Cells from transformant colonies were treated with Zymolyase (ICN Biomedicals; 50 units/ml in 10 mM Tris–HCl/0.1 mM EDTA, pH 7.5, at 37 for 30 min and then at 95 for 10 min) and subjected to junction-specific PCR with primers in the transgenes and flanking endogenous loci to detect correct insertion/deletion mutants (primers and PCR conditions available upon request).