The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance.
In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.
Vanderstraeten
S
S
Unité de Biochimie Physiologique, Place Croix du Sud, 2-20, 1348 Louvain-la-Neuve, Belgium.
Van den Brûle
S
S
Hu
J
J
Foury
F
F
D001483:Base Sequence; D000074002:DNA Polymerase gamma; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000096:biosynthesis / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D016681:Genome, Fungal; D007700:Kinetics; D008938:Mitosis; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D013045:Species Specificity; D013698:Templates, Genetic
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V