This year, we published the following accomplishments. (1) We completed four structure-function studies of DNA synthesis by DNA polymerase lambda, an enzyme that participates in repairing DNA lesions resulting from exposure to environmental stress, including radiation. This includes a study of inhibition of Pol lambda by anticancer nucleotide analogs, and the identification of a polymorphic variant of Pol lambda that decreased genome stability and may be associated with increased cancer susceptibility. (2) We published a comprehensive review of DNA replication infidelity in a symposium volume celebrating Darwins birth and publication of the Origin of Species. (3) We published two articles demonstrating that the major replicative DNA polymerases incorporate ribonucleotides during replication in vitro, that one of these (Pol epsilon) incorporates ribonucleotides during replication in yeast, that these ribonucleotides are normally removed by a RNase H2-dependent repair process, and that failure to repair these ribonucleotides results in genome instability via DNA strand misalignments. (4) We contributed one set of experiments to an article from the Elledge group that discovered and characterized a nuclease involved in the repair of interstrand crosslinks. (5) We completed two studies of DNA polymerase nu, a recently discovered human polymerase of unknown function. In one study, we collaborated with the Petrovich group to re-fold Pol nu from inclusion bodies, a novel approach that should facilitate future studies of this enzyme and may be useful for other proteins that are problematic to express in soluble form. In the other study, we collaborated with the Wood group to characterize mutant derivatives of Pol nu, as one step to eventually understand its function and possible contribution to genome stability

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2010
Total Cost
$72,251
Indirect Cost
City
State
Country
Zip Code
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 :
Kunkel, Thomas A (2018) A simple but profound mutation in mouse DNA polymerase ? drives tumorigenesis. J Clin Invest :
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
Kunkel, Thomas A; Burgers, Peter M J (2017) Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols ?, ?, and ? in the replisome for leading-strand and lagging-strand DNA replication. Bioessays 39:
Williams, Jessica S; Gehle, Daniel B; Kunkel, Thomas A (2017) The role of RNase H2 in processing ribonucleotides incorporated during DNA replication. DNA Repair (Amst) 53:52-58
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
Burgers, Peter M J; Gordenin, Dmitry; Kunkel, Thomas A (2016) Who Is Leading the Replication Fork, Pol ? or Pol ?? Mol Cell 61:492-493

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