title: Antimutator alleles of yeast DNA polymerase gamma modulate the balance between DNA synthesis and excision.
abstract: 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.
authors: Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium. francoise.foury@uclouvain.be
authors: Françoise
authors: F
authors: Foury
authors: Karolina
authors: K
authors: Szczepanowska
mesh_terms: D000483:Alleles; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000235:genetics*; D059645:Mutation Rate; D010641:Phenotype; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D029701:Saccharomyces cerevisiae Proteins; D004256:DNA Polymerase I; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C068352:MIP1 protein, S cerevisiae; D005092:Exonucleases