of Work: The long-term goal of this project is to understand DNA replication fidelity by multiprotein replication complexes. This year, progress was in three areas. We investigated the roles of mismatch repair proteins in the cisplatin and oxaliplatin resistance of human tumor cell lines by examining the correlation between defects in hMLH1, hMSH3, and hMSH6 and replicative bypass of platinum-DNA adducts. The results support the hypothesis that defects in MutS alpha function, but not MutS beta function, lead to increased replicative bypass of cisplatin adducts and therefore drug resistance. We began a search for synergistic survival and mutator effects resulting from mutations in the genes encoding replicative DNA polymerase delta and the flap endonuclease responsible for processing Okazaki fragments on the lagging strand. We also initiated studies to examine the in vivo effects of a specific amino acid substitution in yeast replicative DNA polymerases. We replaced a tryrosine hypothesized to occupy a structurally and functionally equivalent position on an alpha helix at the active site of DNA polymerases in the pol I and Pol alpha family enzymes with an alanine. Our earlier studies showed that a Tyr to Ala substitution in Klenow fragment Pol I resulted in a polymerase with reduced fidelity. We now find that a haploid yeast strain with the putative homologous pol epsilon mutation is viable. It has altered sensitivity to hydroxyurea (replication inhibitor) and methylmethane sulfonate (a DNA damaging agent), and has an elevated spontaneous mutation rate as well as increased mutagenesis induced by hydroxylaminopurine, a base analog with miscoding potential. These phenotypes are under further investigation and will be complemented by attempts to purify and characterize the mutant pol epsilon. These studies of how genomes are efficiently and correctly replicated are important for human health because spontaneous and DNA damage-induced replication errors are likely sources of mutations that may initiate human diseases. - Accessory Proteins, DNA Replication Fidelity, Deletions, Mutator, T4 DNA Polymerase
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