This year our group, often in collaboration with others, published on 10 aspects of DNA synthesis fidelity. (1) We determined the kinetics of Pol nu insertion, misinsertion and mismatch extension in several sequence contexts. We found that the low fidelity of this error-prone human enzyme is primarily due to low catalytic efficiency for inserting correct dCTP rather than high efficiency for misinserting dTTP. (2) We tested whether the roles of Pols delta and epsilon in replication in budding yeast are evolutionarily conserved in fission yeast. Overall, the data indicate that this is the case (3) We tested the hypothesis that Pol zeta participates in SHM. Two mouse models of Pol z function were generated: a B-cell specific conditional knock-out strain and a knock-in strain encoding L2610F Pol z. Pol z-deficient B-cells had reduced mutation frequency at immunoglobulin loci, whereas L2610F mice had markedly increased mutation frequency. The data indicate a direct role for Pol z in SHM. (4) We collaborated to show that accumulation of rNMPs in the yeast genome in the absence of both RNase H1 and RNase H2 causes replication stress and toxicity. Both MMS2-dependent template switching and Pol z-dependent bypass have roles in overcoming these deleterious effects of rNTP misincorporation during DNA replication. (5) We collaborated with Robert E. London to describe the solution structure of the Dickerson DNA dodecamer containing a single ribonucleotide using NMR. (6) We investigated how rNTP incorporation occurs during DNA synthesis by Pol lambda. These structure-function studies improved an understanding of how polymerases use ribo-containing substrates for DNA synthesis, which may be relevant for repair synthesis occurring in non-proliferating cells when rNTP:dNTP ratios are high. (7) We investigated the ability of the exonuclease activity of yeast Pol epsilon to proofread newly inserted ribonucleotides. The results indicate that Pol epsilon does proofread newly inserted rNMPs to enhance genome stability, but proofreading of an incorrect sugar is substantially less efficient than is proofreading of an incorrect base. (8) We contributed to a study describing a complete biochemical reconstitution of the ribonucleotide excision repair pathway with enzymes purified from budding yeast. (9) We determined the specificity of mutations generated in vivo when yeast Pol zeta bypasses spontaneous lesions in the yeast genome. The results indicate that when Pol zeta performs mutagenic bypass of endogenous, helix-distorting lesions, it can perform a short track of processive, error-prone synthesis to generate a truly amazing array of tandem double and clustered mutations. This property may be relevant to multistage carcinogenesis and the evolutionary conservation of Pol zeta. The results also suggest that tandem base pair substitutions and clusters of multiple, closely spaced mutations may be useful biomarkers to indicate a role for TLS by Pol zeta in environmental mutagenesis underlying tumor formation in humans. (10) In collaboration with K. Garrish and P. Bushel, we compared mRNA expression in a wild type yeast strain to that in a rhn201∆strain. Deleting RNH201 alters RNA expression of 349 genes by 1.5-fold (q-value <0.01), of which 123 are up regulated and 226 are down regulated. Differentially expressed genes (DEGs, on right) include those involved in stress responses and genome maintenance, consistent with a role of RNase H2 in removing ribonucleotides incorporated into DNA during replication. Up-regulated genes include several that encode subunits of RNA polymerase I and III, and many involved in ribosomal RNA processing and ribosomal biogenesis and in tRNA modification and processing, supporting a role for RNase H2 in resolving R-loops formed during transcription of rRNA and tRNA genes. Several DEGs are involved in telomere maintenance, supporting a role for RNase H2 in resolving RNA-DNA hybrids formed at telomeres. A large number of DEGs encode nucleases, helicases and genes involved in response to dsRNA viruses, observations that could be relevant to the nucleic acid species that elicit an innate immune response in RNase H2-defective humans.

Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2012
Total Cost
$831,865
Indirect Cost
City
State
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Williams, Jessica S; Lujan, Scott A; Kunkel, Thomas A (2016) Processing ribonucleotides incorporated during eukaryotic DNA replication. Nat Rev Mol Cell Biol 17:350-63
Lujan, Scott A; Williams, Jessica S; Kunkel, Thomas A (2016) Eukaryotic genome instability in light of asymmetric DNA replication. Crit Rev Biochem Mol Biol 51:43-52
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
Lujan, Scott A; Williams, Jessica S; Kunkel, Thomas A (2016) DNA Polymerases Divide the Labor of Genome Replication. Trends Cell Biol 26:640-54
Burgers, Peter M J; Gordenin, Dmitry; Kunkel, Thomas A (2016) Who Is Leading the Replication Fork, Pol ε or Pol δ? Mol Cell 61:492-3
Van, Christopher; Williams, Jessica S; Kunkel, Thomas A et al. (2015) Deposition of histone H2A.Z by the SWR-C remodeling enzyme prevents genome instability. DNA Repair (Amst) 25:9-14
Williams, Jessica S; Clausen, Anders R; Lujan, Scott A et al. (2015) Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific. Nat Struct Mol Biol 22:291-7
Moon, Andrea F; Gosavi, Rajendrakumar A; Kunkel, Thomas A et al. (2015) Creative template-dependent synthesis by human polymerase mu. Proc Natl Acad Sci U S A 112:E4530-6
Miyabe, Izumi; Mizuno, Ken'Ichi; Keszthelyi, Andrea et al. (2015) Polymerase δ replicates both strands after homologous recombination-dependent fork restart. Nat Struct Mol Biol 22:932-8
St Charles, Jordan A; Liberti, Sascha E; Williams, Jessica S et al. (2015) Quantifying the contributions of base selectivity, proofreading and mismatch repair to nuclear DNA replication in Saccharomyces cerevisiae. DNA Repair (Amst) 31:41-51

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