This paper summarizes recent advances in understanding the links between the cell's ability to maintain integrity of its mitochondrial genome and mitochondrial genetic diseases. Human mitochondrial DNA is replicated by the two-subunit DNA polγ- merase γ (pol γ). We investigated the fidelity of DNA replication by pol γ with and without exonucleolytic proofreading and its p55 accessory subunit. Pol γ has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Progressive external ophthalmoplegia (PEO) is a rare disease characterized by the accumulation of large deletions in mitochondrial DNA. Recently, several mutations in the polymerase and exonuclease domains of the human pol γ have been shown to be associated with PEO. We are analyzing the effect of these mutations on the human pol γ enzyme. In particular, three autosomal dominant mutations alter amino acids located within polymerase motif B of pol γ. These residues are highly conserved among family A DNA polymerases, which include T7 DNA polymerase and E. coli pol I. These PEO mutations have been generated in pol γ to analyze their effects on overall polymerase function as well as the effects on the fidelity of DNA synthesis. One mutation in particular, Y955C, was found in several families throughout Europe, including one Belgian family and five unrelated Italian families. The Y955C mutant pol γ retains a wild-type catalytic rate but suffers a 45-fold decrease in apparent binding affinity for the incoming dNTP. The Y955C derivative is also much less accurate than is wild-type pol γ, with error rates for certain mismatches elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C pol γ is consistent with the accumulation of mtDNA mutations in patients with PEO. The effects of other pol γ mutations associated with PEO are discussed.
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