Fidelity of the Human Mitochondrial DNA Polymerase

Harold R Lee,Kenneth A Johnson

doi:10.1074/jbc.m607964200

Harold R Lee, Kenneth A Johnson

Open Access

https://doi.org/10.1074/jbc.m607964200

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Abstract

We have quantified the fidelity of polymerization of DNA by human mitochondrial DNA polymerase using synthetic DNA oligonucleotides and recombinant holoenzyme and examining each of the possible 16-base pair combinations. Although the kinetics of incorporation for all correct nucleotides are similar, with an average Kd of 0.8 microM and an average k(pol) of 37 s(-1), the kinetics of misincorporation vary widely. The ground state binding Kd of incorrect bases ranges from a low of 25 microM for a dATP:A mispair to a high of 360 microM for a dCTP:T mispair. Similarly, the rates of incorporation of incorrect bases vary from 0.0031 s(-1) for a dCTP:C mispair to 1.16 s(-1) for a dGTP:T mispair. Due to the variability in the kinetic parameters for misincorporation, the estimates of fidelity range from 1 error in 3563 nucleotides for dGTP:T to 1 error in 2.3 x 10(6) nucleotides for dCTP:C. Interestingly, the discrimination against a dGTP:T mismatch is 16.5 times lower than that of a dTTP:G mismatch due to a tighter Kd for ground state binding and a faster rate of incorporation of the dGTP:T mismatch relative to the dTTP:G mismatch. We calculate an average fidelity of 1 error in 440,000 nucleotides.

Highlights

Administration of nucleoside analogs as part of the highly active antiretroviral therapy to combat human immunodeficiency virus infections [10] causes duration-dependent mitochondrial dysfunction
Previous analysis of the discrimination against nucleoside analog reverse transcriptase inhibitors (NRTIs) by human mitochondrial DNA polymerase (Pol ␥)2 has defined a toxicity index based upon the increased time required to replicate the mitochondrial genome when NRTIs are present [14, 15]
Exonuclease-deficient Pol ␥ was used for these misincorporation experiments because neither creation of a mismatch nor extension beyond a mismatch have been observed in the presence of exonuclease function [23, 28, 29]

Summary

Fidelity of the Human Mitochondrial DNA Polymerase*

Previous analysis of the discrimination against nucleoside analog reverse transcriptase inhibitors (NRTIs) by human mitochondrial DNA polymerase (Pol ␥) has defined a toxicity index based upon the increased time required to replicate the mitochondrial genome when NRTIs are present [14, 15]. A study using another Pol A family enzyme, the Klenow fragment of DNA Pol I, showed that of all of the 12 possible incorrect pairings, a dGTP incorporated onto a template dTMP was the most common mutation [22]. Single nucleotide incorporation assays showed that the fidelity of Klenow fragment manifests from a ϳ25-fold increase in the Kd for ground state binding and an ϳ3000-fold decrease in the rate of incorporation on the average. Pol ␥, human mitochondrial DNA polymerase holoenzyme; BF, Bacillus DNA polymerase I fragment; Pur, purine; Pyr, pyrimidine In the accompanying paper we examine the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine triphosphate, a common oxidative metabolite of dGTP [27]

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Specificity Discrimination