Structure-Function Studies Of DNA Replication Fidelity

Environmental Health Sciences

Investigators

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Abstract

This year, sometimes with the group only and other times in productive collaborations with scientists in other groups within NIH and elsewhere, we had several accomplishments. (1) We determined the effects of three replication accessory protein complexes, RPA, RFC and PCNA, on the fidelity of DNA synthesis by the major replicative DNA polymerase, pol delta (B family). (2) We determined the fidelity of DNA synthesis by yeast DNA polymerase zeta (B family), which has a critical role in determining the rate of both spontaneous and DNA damage-induced mutagenesis in eukaryotes. (3) We determined the efficiency and fidelity of translesion DNA synthesis (TLS) by a highly processive and normally highly accurate model replicative DNA polymerase, T7 pol (A family), for which there is available elegant structural information. (4) We constructed and characterized mouse models deficient in one or both of two mammalian Y family DNA polymerases, pol eta and pol iota. The results establish a clear role for pol iota in TLS and in prevention of skin cancer. (5) We determined the fidelity of DNA synthesis by (A family) human DNA polymerase nu. Although its biological function is unknown, this enzyme has low fidelity and unusual error specificity, suggesting a highly specialized function. (6) We determined the X ray crystal structure and perform very informative studies of the substrate specificity of DNA polymerase mu, an X family polymerase involved in repairing double strand breaks in DNA by a non-homologous end-joining pathway. (7) We identified and thoroughly characterized the biochemical properties of a mutant derivative of the major replicative DNA polymerase, pol delta (B family). (8) We performed biochemical studies of the mutagenic potential of a common lesion in DNA generated by oxidative stress, 2-OH-adenine. (9) We performed studies indicating that RNA can serve as a template for DNA repair synthesis in yeast. (10) We performed experiments indicating that DNA polymerase epsilon (B family) participates in replicating the leading strand template of the yeast nuclear genome. (11) We solved the X ray crystal structure of a ternary complex of DNA polymerase lambda (X family) with a non-hydrolyzable nucleotide triphosphate that provided important insights into the catalytic mechanism for polymerization.. (12) We identified and thoroughly characterized the biochemical properties of mutant derivative of the major replicative DNA polymerase, pol epsilon (B family). (13) We determined the effects of three replication accessory protein complexes, RPA, RFC and PCNA, on the fidelity of TLS by DNA polymerase eta (Y family). (14) We published a comprehensive review of structure-function relationships among the four mammalian X family DNA polymerases. (15) We identified and genetically characterized a mutator derivative of yeast DNA polymerase zeta (B family), which as mentioned above has a critical role in determining the rate of both spontaneous and DNA damage-induced mutagenesis in eukaryotes.