We are using DNA polymerases obtained by recombinant DNA technology to examine the mechanisms and protein-DNA interactions that are important for determining the fidelity of DNA synthesis. We have determined the fidelity of DNA synthesis catalyzed by the normal Klenow polymerase, by two mutant derivatives lacking proofreading exonuclease activity but having a normal protein structure, and by a protein that contains only one of two domains, the large polymerase domain. The fidelity results have permitted the formulation of four models to explain the production of base substitution and frameshift errors. We have been testing each of these. We have also established the fidelity of the thermostable Taq polymerase used in polymerase chain reactions (PCR), using various reaction conditions, including changes in temperature, pH, relative and absolute dNTP con- centration and Magnesium Chloride, concentration. These studies define high fidelity conditions that are useful for genetic applications of DNA amplified by PCR. We are currently examining mutant derivatives of three recombinant DNA polymerases for which structural information is available, (E. coli DNA polymerase I, HIV-1 reverse transcriptase DNA polymerase beta.) It is our belief that extensive analyses of these enzymes offers the best hope for understanding the molecular determinants for accurate DNA synthesis as well as how this process can be compromised by DNA adducts.
| 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 |
| 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 |
| 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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