DNA REPLICATION FIDELITY

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Summary 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 spent considerable effort to write and publish an extensive review on the fidelity of DNA replication as interpreted from evolving structural information on DNA polymerases. We successfully established a model system that will allow investigation of the fidelity of base excision repair. We determined that repair of a GoU mismatch that is catalyzed by four highly purified recombinant human proteins works well and indicates that repair fidelity is not high. We are now investigating hypotheses that could explain genome stability in light of this new information. We also continued studies to examine the effects on genome stability in vivo of amino acid substitutions in the putative active sites of five different yeast DNA polymerases. Alanines were substituted for tryrosines hypothesized to occupy positions that are structurally and functionally equivalent to a tyrosine on an alpha helix at the active site of DNA polymerase I that is critical for replication fidelity. Interesting phenotypes have been obtained with the yeast polymerase mutants, including either enhanced or reduced genome stability and altered sensitivity to DNA damaging agents. These phenotypes depend on which of five polymerases is modified. Further investigation of these phenotypes is in progress, and will be complemented by attempts to purify and characterize the mutant polymerases. 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.