of Work: The long-term goal of this project is to understand the fidelity of DNA synthesis by multiprotein DNA replication and repair complexes. This year, progress was made in three areas. 1) We provided evidence that pol eta lacks an intrinsic proofreading exonuclease and copies undamaged DNA with very low fidelity, indicating a relaxed requirement for correct base pairing geometry and suggests that the function of pol eta may be tightly controlled to prevent potentially mutagenic DNA synthesis in human cells. This year we tested this hypothesis and obtained evidence in vitro and in vivo that pol eta errors can be corrected by an extrinsic proofreading exonuclease and by DNA mismatch repair. 2) We collaborated to demonstrate that the intrinsic proofreading exonuclease of the major replicative DNA polymerase delta has a second important role in vivo, the processing the 5' ends of Okazaki fragments to prevent lethal double strand DNA breaks. 3) We determined the effects on genome stability in vivo of amino acid substitutions in the putative active sites of all yeast family B DNA polymerases. Alanine was substituted for two different tyrosines hypothesized to occupy positions that are structurally and functionally equivalent to tyrosines in the active site of E. coli DNA polymerase I that is critical for replication fidelity. Interesting phenotypes were obtained, including either enhanced or reduced genome stability and altered sensitivity to DNA damaging agents. These phenotypes depend on which of five polymerases is modified. These studies of how genomes are efficiently and correctly replicated and repaired are important for human health because spontaneous and DNA damage-induced replication errors are likely sources of mutations that may initiate human diseases.
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