Contributions of DNA polymerase delta proofreading to replication fidelity in Saccharomyces Cerevisiae

Each time a cell divides it must create an accurate copy of its genetic information. Faithful DNA replication is achieved by a series of overlapping mechanisms. The DNA polymerase selectively inserts nucleotides into the growing strand of DNA. Occasional misinsertions are extended slowly, creating the opportunity for removal by 3′ to 5′ exonucleolytic proofreading. Replication errors that escape proofreading are subject to mismatch repair. This research has focused on eukaryotic proofreading and employs the simple model eukaryote, S. cerevisiae. Mutational analysis of the exonuclease (Exo) domain of one of the main replicative polymerases in yeast, polymerase δ (Pol δ), highlights the importance of the conserved Exo carboxylates to proofreading function. Two Exo subdomains were identified as important to replication fidelity by screening alanine-scanning mutants for mutator phenotypes. This research describes the cooperative relationship between Pol δ proofreading and Msh6-mediated mismatch repair in repairing polymerase errors and utilizes this relationship to uncover weak Pol δ mutator alleles. An inverse correlation was noted between mutation rate and cell growth; the most mild msh6Δ Pol δ mutator cells grow normally; msh6Δ moderate Pol δ mutator cells grow slowly; and the strongest msh6Δ Pol δ mutator cells do not grow. One explanation of these data is that the cells are experiencing error catastrophe, a phenomena in which cells die by lethal mutagenesis. Examination of rare, growing msh6Δ strong Pol δ mutator colonies led to the discovery of second site mutations in Pol δ that restore growth to msh6Δ strong Pol δ mutator cells and reduce the mutation rate of MSH6 + strong Pol δ mutator cells. The rescue of growth in strong mutator strains by antimutator alleles lends further support to the notion that msh6Δ strong Pol δ mutator yeast strains are experiencing error catastrophe, and highlights the vital role of polymerase proofreading in DNA replication fidelity.