| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 99 === DNA polymerases play a central role in DNA replication and DNA repair upon damage. Here we describe two structurally similar DNA polymerases involved in DNA base excision repair (BER) pathway, which is crucially important in maintaining genetic integrity. (1) DNA polymerase β (Pol β) is a high fidelity DNA polymerase, and serves as an ideal kinetic model for studying the mechanism of nucleotidyl transfer catalysis. By stopped flow, we proposed a hypothesis for the Mg2+ inhibition mechanism from [Mg2+] dependence and R258A mutant studies. From measuring proton formation by pH indicator, we provide direct evidence that the rate-limiting step during catalysis at high pH is the subdomain re-opening after chemistry. (2) African swine fever virus (ASFV) DNA polymerase X (Pol X) is distinct in that it is considered to be low fidelity that involves specific catalysis of dGTP insertion toward DNA template dG, which may be important in the strategic mutagenesis of the viral genome. Our NMR study has implicated the importance of His115 for Pol X nucleotide specificity in G:G mismatch, thus the binding constants for Pol X-dNTP complexes were determined by isothermal titration calorimetry (ITC). Free Pol X shows distinct nucleotide specificity on dGTP, but in DNA with template dG, the formation of the dCTP ternary complex is favored instead. The H115A mutation significantly weakens nucleotide binding, and impairs catalysis of both G:G and G:C insertion. These results provide another example to support previous hypothesis of the error prone Pol X on facilitating strategic mutagenesis in ASFV.
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