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Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase eta (yPoleta), 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 yPoleta which contains only the polymerase domain. In the absence of yPoleta'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 yPoleta may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPoleta 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.
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