Fidelity of Nucleotide Incorporation by Human Mitochondrial DNA Polymerase

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Abstract
We have examined the fidelity of polymerization catalyzed by the human mitochondrial DNA polymerase using wild-type and exonuclease-deficient (E200A mutation) forms of recombinant, reconstituted holoenzyme. Each of the four nucleotides bind and incorporate with similar kinetics; the average dissociation constant for ground state binding is 0.8 μm, and the average rate of polymerization is 37 s−1, defining a specificity constant kcat/Km = 4.6 × 107m−1 s−1. Mismatched nucleotides show weaker ground-state nucleotide binding affinities ranging from 57 to 364 μm and slower rates of polymerization ranging from 0.013 to 1.16 s−1. The kinetic parameters yield fidelity estimates of 1 error out of 260,000 nucleotides for a T:T mismatch, 3563 for G:T, and 570,000 for C:T. The accessory subunit increases fidelity 14-fold by facilitating both ground-state binding and the incorporation rate of the correct A:T base pair compared with a T:T mismatch. Correctly base-paired DNA dissociates from the polymerase at a rate of 0.02 s−1promoting processive polymerization. Thus, the mitochondrial DNA polymerase catalyzed incorporation with an average processivity of 1850, defined by the ratio of polymerization rate to the dissociation rate (37/0.02) and with an average fidelity of one error in 280,000 base pairs.