antimutator alleles of yeast dna polymerase gamma modulate the balance between dna synthesis and excisionantimutator等位基因的酵母dna聚合酶伽马调节dna合成之间的平衡和切除.pdfVIP

Antimutator Alleles of Yeast DNA Polymerase Gamma Modulate the Balance between DNA Synthesis and Excision Franc¸oise Foury*, Karolina Szczepanowska ´ Institut des Sciences de la Vie, Universite Catholique de Louvain, Louvain-la-Neuve, Belgium Abstract Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol c ), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 39-59 exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol c (Mip1). These mip1 mutations, localised in the 39-59 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 synth