DNA polymerases ␦ and ⑀ (pol ␦ and ⑀) are the major replicative polymerases and possess 3-5 proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol ⑀, the 3-5 exonuclease-deficient pol2-4, a ؉1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol ⑀ was 0.47 ؋ 10 ؊5for G:G mismatches, 0.15 ؋ 10 ؊5 for T:G mismatches, and less than 0.01 ؋ 10 ؊5 for A:G mismatches. The accuracy for A opposite G was not altered in the exonucleasedeficient pol2-4 pol ⑀; however, G:G and T:G misincorporation rates increased 40-and 73-fold, respectively. The pol2C1089Y pol ⑀ mutant also exhibited increased G:G and T:G misincorporation rates, 22-and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3-5 exonuclease activity of pol2C1089Y pol ⑀ is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.The yeast Saccharomyces cerevisiae has three DNA polymerases, (pol ␣, ␦, and ⑀), which are required for cell growth, chromosomal DNA replication (1), and DNA double-strand break repair (2). pol ␣ has four subunits (Pol1 (Cdc17), Pol10, Pri1, and Pri2) and is involved primarily in the initiation of DNA replication and priming of Okazaki fragments. pol ␦ and pol ⑀ are required during synthesis of the leading and lagging DNA strands at the replication fork; they bind at/or near replication origins and move along DNA with the replication fork (3, 4). The precise roles of pol ␦ and pol ⑀ during leading and lagging strand DNA synthesis have not yet been defined; however, genetic and biochemical evidence suggest that lagging strand DNA synthesis is carried out by pol ␣ and pol ␦ (5, 6).pol ␦ of S. cerevisiae has three subunits (Pol3 (Cdc2), Hys2 (Pol31) (7,8), and Pol32 (8)), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1, and Cdc27, respectively (9). S. pombe pol ␦ has one additional subunit, Cmt1 (9). Purified yeast pol ␦ requires accessory factors including PCNA 1 and RFC to catalyze processive DNA synthesis (8). pol ␦ possesses a 3Ј-5Ј exonuclease activity, which performs proofreading/editing during DNA synthesis (10, 11).The S. cerevisiae pol ⑀ is also a multi-subunit protein complex that includes Pol2, Dpb2, Dpb3, and Dpb4 and like pol ␦ has a 3Ј-5Ј exonuclease activity (1, 12). pol ⑀ is a highly processive enzyme (12)(13)(14). Although pol ⑀ requires PCNA and RFC complex to catalyze processive DNA synthesis on singly primed single-stranded viral DNA (13), these cofactors may not be required for processive DNA synthesis in vivo. Pol2 is the catalytic subunit of pol ⑀ and is encoded by the POL2 gene (15), which is essential for cell growth and re...

The DNA polymerases δ and ε are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3′ → 5′ 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 +1 frameshifts while there was almost no effect on −1 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 +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the −1 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes +1 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 +1-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polε.