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 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.
Brown
Jessica A
JA
Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Zhang
Likui
L
Sherrer
Shanen M
SM
Taylor
John-Stephen
JS
Burgers
Peter M J
PM
Suo
Zucai
Z
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