Antimutator Alleles of Yeast DNA Polymerase Gamma Modulate the Balance between DNA Synthesis and Excision

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Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol γ ), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3′-5′ exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol γ (Mip1). These mip1 mutations, localised in the 3′-5′ exonuclease and polymerase domains, elicit a 2–15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol γ area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.