Pre-steady-state kinetic analysis of truncated and full-length Saccharomyces cerevisiae DNA polymerase eta

Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPol η), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPol η which contains only the polymerase domain. In the absence of yPol η 's C-terminal residues 514-632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPol η may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPol η discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide 5′ -triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides.