Unique Error Signature of the Four-subunit Yeast DNA Polymerase ε

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ε (Pol ε) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol ε synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of ≤2 × 10^-5 and ≤5 × 10^-7, respectively. Pol ε lacking 3′ → 5′ exonuclease activity is less accurate to a degree suggesting that wild type Pol ε proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol ε is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP, and C-dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol ε differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol ε in vitro and substitutions occurring in vivo in a yeast strain defective in Pol ε proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol ε polymerase activity in DNA replication.