We are using mutant enzymes obtained by recombinant DNA technology to examine the relationship between the structural and kinetic properties of DNA polymerases and their fidelity. Emphasis is on polymerases for which X-ray crystal structure information is or may soon become available. We have established the processivity and error specificity of exonuclease-deficient Klenow polymerase at two reaction pHs. We extended the anlaysis to three mutants in which a tyrosine residue at position 766, in the """"""""O helix"""""""" of the polymerase active site, was changed to an alanine, serine or phenylalanine, and to additional active site residues involved in catalysis and metal binding. Several of these mutants have altered processivity and altered fidelity, some having mutator and others antimutator phenotypes. The data suggest that dNTP and metal binding residues are important for determining base selectivity, but (to date) not template-primer misalignment-initiated errors. We are presently examining mutant derivatives of the HIV-1 reverse transcriptase, in which we have mutated the """"""""thumb"""""""" residues known to be important for binding double-stranded template-primer. We have also begun to establish the fidelity of the wild-type T4 and T7 DNA polymerase, as well as their exonuclease-deficient partners differing by one and two conserved amino acids, respectively. Analyses of recombinant DNA polymerases for which considerable genetic and/or kinetic data are available should improve our understanding of accurate DNA synthesis and how fidelity is affected by DNA adducts.
| Kaminski, Andrea M; Tumbale, Percy P; Schellenberg, Matthew J et al. (2018) Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis. Nat Commun 9:2642 |
| Orebaugh, Clinton D; Lujan, Scott A; Burkholder, Adam B et al. (2018) Mapping Ribonucleotides Incorporated into DNA by Hydrolytic End-Sequencing. Methods Mol Biol 1672:329-345 |
| Burkholder, Adam B; Lujan, Scott A; Lavender, Christopher A et al. (2018) Muver, a computational framework for accurately calling accumulated mutations. BMC Genomics 19:345 |
| Zhou, Zhi-Xiong; Williams, Jessica S; Kunkel, Thomas A (2018) Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis. J Vis Exp : |
| Williams, Jessica S; Kunkel, Thomas A (2018) Studying Topoisomerase 1-Mediated Damage at Genomic Ribonucleotides. Methods Mol Biol 1703:241-257 |
| Huang, Shar-Yin N; Williams, Jessica S; Arana, Mercedes E et al. (2017) Topoisomerase I-mediated cleavage at unrepaired ribonucleotides generates DNA double-strand breaks. EMBO J 36:361-373 |
| Jamsen, Joonas A; Beard, William A; Pedersen, Lars C et al. (2017) Time-lapse crystallography snapshots of a double-strand break repair polymerase in action. Nat Commun 8:253 |
| Burgers, Peter M J; Kunkel, Thomas A (2017) Eukaryotic DNA Replication Fork. Annu Rev Biochem 86:417-438 |
| Lujan, Scott A; Williams, Jessica S; Kunkel, Thomas A (2016) DNA Polymerases Divide the Labor of Genome Replication. Trends Cell Biol 26:640-654 |
| Watt, Danielle L; Buckland, Robert J; Lujan, Scott A et al. (2016) Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools. Nucleic Acids Res 44:1669-80 |
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