Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations
© Pavlov et al; licensee BioMed Central Ltd. 2004
Received: 16 January 2004
Accepted: 26 May 2004
Published: 26 May 2004
DNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect.
Purified four-subunit Y831A Pol ε turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol ε, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol ε, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol δ and Pol α suggest that Pol ε may proofread certain errors made by Pol α and Pol δ during replication in vivo.
Our data suggest that Y831A replacement in Pol ε reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase ε.
Multiple DNA polymerases are thought to be present at the eukaryotic replication fork [1–4]. Some of their functions could be unique while others could be overlapping. Different polymerases may compete for certain DNA substrates and several polymerases may sometimes act in concert [5–9]. Under normal circumstances, chromosomal replication requires at least three DNA polymerases, Pol α, Pol ε and Pol δ. All of these polymerases are multi-subunit complexes [1, 4] and all subunits are required for their proper function (see recent papers [10–12], and references therein). Pol α is not very processive and lacks an intrinsic proofreading exonuclease. It has a tightly associated activity for the synthesis of RNA primers at replication origins and on the lagging DNA strand. Pol α extends these RNA primers by synthesizing short stretches of DNA, and then a switch occurs to processive synthesis by Pol ε and/or Pol δ.
The exact roles of Pol ε and Pol δ in replication are not yet fully understood. Among several possible models, it has been proposed that Pol ε is primarily responsible for copying the leading strand DNA template and Pol δ is responsible for lagging strand replication . Another model has proposed the opposite . Either model is consistent with the fact that Pol ε and Pol δ both possess intrinsic 3' to 5' exonuclease activity, and with genetic data in yeast suggesting that these nucleases proofread replication errors on opposite DNA strands during chromosomal  or plasmid  DNA replication. However, the replication functions of Pol δ and Pol ε are not equivalent . When proofreading or base selectivity is impaired by homologous active site point mutations in POL3 (encoding Pol δ) and POL2 (encoding Pol ε), the mutator effects are much stronger for pol3 mutants than for pol2 mutants [7, 18, 19]. A yeast strain with an amino-terminal deletion of the polymerase domain of the POL2 gene but retaining the carboxyl-terminal domain grows slowly but is nonetheless viable [20, 21], and references therein), indicating that another polymerase can substitute for the polymerization function of Pol ε. Thus, it is possible that Pol δ may perform the bulk of chain elongation during chromosomal replication , while Pol ε serves more specialized roles. One possibility is a role in the S phase checkpoint control when replication fork progression is impeded , perhaps by sensing single-stranded DNA . Pol ε also interacts with Dpb11 and, thus, may function during initiation of DNA replication at origins [24, 25]. It has also been suggested that Pol ε may participate in replication during late, but not early, S phase , as well as in the establishment of sister chromatid cohesion .
The fidelity of nuclear DNA replication in eukaryotic cells relies on three steps that are thought to operate in series: the base selectivity of DNA polymerases that operate at the fork, proofreading by the exonucleases of DNA polymerases ε and δ or possibly by extrinsic exonucleases, and DNA mismatch repair. This is supported genetically by the fact that the double mutants that are deficient in proofreading by either DNA polymerase and mismatch repair are hypermutable, suggesting a sequential action of proofreading and mismatch repair [19, 28]. As one attempt to understand the complex enzymology that determines fidelity and influences eukaryotic genome stability, we recently described the effects on viability and mutagenesis in Saccharomyces cereivisiae resulting from replacing a conserved tyrosine in the active sites of three replicative polymerases with alanine . Important for our understanding of the role of Pol ε, a haploid yeast strain with this pol2-Y831A mutation grew normally, suggesting no major defects in replication. However, we observed a modest, spontaneous base substitution mutator effect and a strong, spontaneous frameshift mutator effect when DNA mismatch repair was nonfunctional. These mutator effects suggested a defect in replication fidelity at a step preceding mismatch repair (for example, reduced polymerase base selectivity and/or proofreading) . The pol2-Y831A mutation was semi-dominant in heterozygous diploid cells, suggesting that the mutator effects reflected an aberrant function of the altered Pol ε that is not masked by the presence of wild-type Pol ε. Here we further investigate the mutator effects conferred by the pol2-Y831A mutation using biochemical and genetic approaches. The results suggest decreased participation of Y831A Pol ε in chromosomal replication. We also investigate the possibility that wild-type Pol ε proofreads replication errors generated by altered variants of DNA polymerases α and δ.
Biochemical characterization of Y831A Pol ε
Mutation rates in haploids with double pol2-Y831,4mutation
Effect of combining the exonuclease and active site mutations in Pol ε gene on mutation rates in haploids in 8C-YUNI101 genetic background
Mutation rates for different markers*
Canr (× 10-7)
Ura+ × (10-8)
His+ × (10-8)
Mismatch repair proficient strains
Mismatch repair deficient strains (pms1)
Effect of combining the exonuclease and active site mutations in DNA polymerase ε on mutation rates in diploids. Mismatch repair proficient strains
His+ reversion rate (× 10-8)
Effect of combining the exonuclease and active site mutations in DNA polymerase ε on mutation rates in diploids. Mismatch repair deficient strains (pms1/pms1)
His+ reversion rate (× 10-8)
Lack of genetic interaction between pol2-Y831A and pol3-01mutation in exonuclease domain of Pol δ
Effect of combining the mutation in the exonuclease domain of the Pol δ gene with the active site mutation in Pol ε genes on mutation rates in mismatch repair-proficient derivatives of 8C-YUNI101 strain
Mutation rates for different markers
Canr × (10-7)
Base substitutions Ura+ (× 10-8)
Frameshifts (+1) His+ (× 10-8)
Genetic interaction between active site mutations in genes encoding Pol α and Pol δ and proofreading defective mutations in POL2 and POL3
Effect of combining the mutation in exonuclease domains of DNA polymerases δ and ε genes with active site mutation in DNA polymerases α and δ genes on mutation rates in mismatch repair proficient haploid derivatives of 8C-YUNI101
Forward Canr mutation
Ura+ reversion (base substitutions)
His+ reversion (+ 1 frameshifts)
Rate (× 10-7)
Rate (× 10-8)
Rate (× 10-8)
Investigation of the mechanism of the mutator phenotype of the yeast Y831A DNA Pol ε mutant began with the observation that, for an equivalent amount of polymerization activity, the purified Y831A enzyme has an increased ability to convert dNTPs to dNMPs relative to wild-type Pol ε (Figure 2). The replacement of alanine for a tyrosine in the polymerase active site that is conserved in the B family of DNA polymerases alters the relative activities of the polymerase and exonuclease active sites of Pol ε. The mutator effect of such mutations is unexpected, since in classical studies with T4 DNA polymerase, mutants that lead to increased nucleotide turnover were antimutators (see ).
As the Y831A amino acid replacement is in the polymerase active site rather than the exonuclease active site, it seems likely that the altered protein is somehow compromised in its ability to extend primers, perhaps reflecting reduced catalytic efficiency or increased dissociation from DNA. Any polymerization defect resulting from the alanine substitution is apparently not severe enough to affect the essential role of Pol ε in cell viability, because a haploid yeast strain containing the pol2-Y831A mutation has normal growth characteristics . This viability provided the opportunity, and the increased dNTP turnover provided the motivation, to investigate further the role of Pol ε and its proofreading activity in vivo by looking at the combined effects of polymerase and exonuclease active site mutations on mutation rates. These experiments lead to several interpretations.
The relative increase in dNTP turnover suggests altered communication between the polymerase and exonuclease. This may be relevant to the frameshift mutagenesis promoted by the pol2-Y831A mutation (Tables 1,2,3,4 and ). For example, misaligned intermediates may form as the primer partitions between double-stranded DNA in the polymerase active site and single-stranded DNA in the exonuclease active site (for example, see [34–36]).
An alternative model is based on the hypothesis that Pol ε has three distinct roles in replication (Figure 3B). We propose that wild-type Pol ε has a limited DNA synthetic role during S-phase (Figure 3B, left). Indeed, its in vitro fidelity is distinct from all replicative DNA polymerases studied so far . Since the mutator effect of the pol2-4 is approximately 10-fold lower that that of the pol3-01, we can tentatively estimate that Pol ε would catalyze less than 10% of the bulk DNA synthesis during replication (indicated by thin arrow). This calculation is made under the assumption that Pol ε and Pol δ have similar base selectivity (currently under investigation). However, Pol ε may have more substantial roles in replicating certain regions/sites in the genome (arrow in the center), perhaps where proofreading is less active (for example, long homopolymeric runs, see ), and in performing limited reactions that are important for vegetative growth, for example, initiation of DNA replication ). This hypothesis is consistent with the genetic interactions of the pol2-4 and pol2-Y831A combinations in cis and trans (Tables 1,2,3). In this model, the mutator effect of the pol2-4 allele results from Pol ε involvement in general S-phase replication (Figure 3B, left). The Y831A amino acid change alters the enzyme as discussed above, reducing its involvement in general S-phase replication (indicated by a dashed thin arrow), but permits its functions in the two other pathways, albeit with reduced fidelity (Figure 3B, right). The mutator effects of the pol2-Y831A might then reflect inaccurate replication at specific sites, or general perturbation of replication, rationalizing the lack of synergistic increases in mutation rates at some loci in strains with the pol2-4 (Table 1) or pol3-01 (Table 4) alleles and, on the other hand, semi-dominant mutator effect of pol2-Y831A in diploids when another allele is wild-type POL2. Alternatively or additionally, altered replication efficiency or fidelity in strains harboring mutant polymerase alleles may lead to activation of a checkpoint response that results in the accumulation of mutations .
The model in Figure 3B can also explain the increases in mutagenesis in the pol2-4 pol1-Y869A and pol2-4 pol3-Y708A mutant strains (Table 5), by invoking increased DNA synthesized by Pol ε during S-phase when Pol α or Pol δ is impaired. Especially interesting here are the synergistic increases in mutation rates at the CAN1 locus. This synergy suggests that under certain circumstances, at least some errors generated by Pol α and Pol δ may be proofread by the intrinsic exonuclease activity of Pol ε. In the future, it will be interesting to test this hypothesis further, to determine under what circumstances, for example, translesion DNA synthesis  or base excision repair  errors made by one DNA polymerase may be proofread by an exonuclease intrinsic to a second DNA polymerase or even another protein .
Y831A replacement reduces replication fidelity and participation of Pol ε in chromosomal replication, but without eliminating an additional function of Pol ε that is essential for viability. This suggests that other polymerases can substitute for certain functions of Pol ε. Conversely, Pol ε can proofread errors made by polymerases α and δ.
For mutant construction we used strains 8C-YUNI101 and E134 described earlier . Y831A DNA polymerase ε holoenzyme was purified from the protease-deficient strain ep831-T334 (MATα pol2-Y831A pep4-3 prb1-1122 reg1-501 gal1 ura3-52 leu2-3,112 trp1::hisG can1 LYS2). To construct the strain, we introduced the pol2-Y831A allele into the strain T334 , a Trp- derivative of 334 described in , by a method described earlier .
Construction of mutants with DNA polymerase mutations
All mutations were introduced into chromosomal DNA polymerase genes by the integration-excision method described earlier [7, 18]. Double mutants were constructed sequentially. To construct a double mutation in the POL2 gene, we transformed a strain already possessing pol2-Y831A to Ura+ with the plasmid pJB1 (carrying the pol2-4 mutation) cut by BamHI. Transformants were plated on a medium with 5-fluoroorotic acid to select for Ura- clones resulting from the loss of the plasmid. Resulting clones were tested by PCR for the presence of both mutations. Detection of pol2-Y831A was described previously . To detect the pol2-4 mutation, we amplified a 481 bp region of the POL2 gene using primers M32-m, 5'TCCGAGTATCTATAGACAAGGA and M36, 5'CTCACCTTCAGCATCTGG. The resulting fragment was digested with SfcI. The presence of the pol2-4 mutation creates a new SfcI site; the PCR fragment is cut into 119 and 362 base pair fragments. The same mutations were created in basic haploid strains 8C-YUNI101 and E134. The resulting mutants were then crossed to generate diploids homozygous or heterozygous for polymerase mutations.
Mutation rate measurements
Measurements of mutation rates were performed as described .
Purification of DNA polymerase ε
The four-subunit DNA polymerase ε was purified from 1.2 kg of the protease deficient ep831-T334 strain as described in [23, 29] with modifications. Briefly, after an ammonium sulfate precipitation step and SP-Sepharose chromatography [23, 29], the dialyzed sample was loaded onto a Q Sepharose HP60/100 column (Amersham Biosciences Corp., Piscataway, NJ, USA) and eluted with nine column volumes of a linear gradient of NaCl from 50 to 500 mM. Four peaks with DNA polymerase activity were obtained, and the peak containing the four-subunit Pol ε was identified by immunoblotting with polyclonal antibodies . Further fractionation of pooled fractions from this peak by MonoS H/R and HiTrap heparin columns (Amersham Biosciences) was performed as described . Finally, fractions with DNA polymerase ε from the Heparin column were loaded onto a MonoS 1.6/5 column in a SMART system (Amersham Biosciences). The polymerase was eluted by a linear NaCl gradient from 50 to 1000 mM. Fractions (30 μl) were collected and assayed for activity. One UV absorbance peak was observed and it coincided with one peak of polymerase activity. The purity of the preparation was approximately 90% as judged from SDS-PAGE electrophoresis and SimpleStain (Invitrogen, Carlsbad, California, USA) colloidal coumassie staining (Figure 1). Peak fractions with specific polymerase activity around 4,000 U/mg were pooled and stored at -80°C.
Enzyme activity determination
Polymerase activity was determined using a poly(dA)300/oligo(dT)10 substrate as described . Reaction mixtures (20 μl) were incubated at 30°C, and 5 μl aliquots were withdrawn at the indicated time intervals and placed in tubes with 1 μl of 300 mM EDTA. One μl of these mixtures was spotted onto each of the two PEI (Merck, Whitehouse Station, NJ, USA) plates. One was developed in 1 M LiCl, 1 M formic acid, 50 mM NaH2PO4, thus separating the unincorporated label from label incorporated into DNA. A second plate was developed with essentially the same solution but containing only 0.4 M LiCl. This permits quantification of the amount of labeled nucleoside monophosphate generated (that is, turnover of dNTP by the DNA polymerase-associated proofreading exonuclease, see Figure 2A,2B). The intensity of the spots was quantified by BAS2500 Bio Image Analyzer (Fuji Photo Film Company, Tokyo, Japan).
Exonuclease assays employed a 3'-radiolabelled poly(dA)300 single-stranded substrate. Three μg of poly(dA)300 were labeled by terminal transferase with 40 μCi of α-32P deoxyadenosine triphosphate (dATP) in 1 mM dATP and 1x One-for-All Plus buffer (Promega, Madison, WI, USA). Reaction products were purified using a Qiagen nucleotide removal kit (Qiagen, Valencia, CA, USA) and then examined by TLC. An average of nine dATP molecules were added per each poly(dA)300 molecule. Exonuclease reactions (20 μl) with 37 ng (0.14 μCi) of labeled substrate and 200 ng of unlabeled poly(dA)300 were performed in the same buffer as the polymerase reaction, also at 30°C. At each time point, one μl of reaction mixture was spotted onto a PEI plate and developed with 0.4 M LiCl as described above. The polymerase activity estimate was compensated by dividing the apparent value of incorporation (acid insoluble nucleotides) with 0.78 for the wild-type enzyme (turnover rate = 22%) and 0.50 for the mutant enzyme (turnover rate = 50%).
We would like to thank Drs A Sugino and H Araki for valuable comments throughout the course of this study, and PV Shcherbakova and R Kokoska for critical reading of the manuscript.
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