The Fidelity of Human DNA Polymerase γ with and without Exonucleolytic Proofreading and the p55 Accessory Subunit

Abstract

Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase γ (pol γ), we investigated the fidelity of DNA synthesis by human pol γ. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol γ has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol γ is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying homopolymeric sequences longer than four nucleotides, pol γ has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol γ both to make and to proofread dislocation intermediates is the first such evidence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol γ catalytic subunit, decreases frameshift and base substitution fidelity. Kinetic analyses indicate that p55 promotes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frameshift mutation in vivo due to replication errors by pol γ.