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Papers In Press, published online ahead of print July 26, 2001
J. Biol. Chem, 10.1074/jbc.M106045200
Submitted on June 28, 2001
Revised on July 25, 2001
Accepted on July 25, 2001
Chemistry & Biochemistry, University of Texas at Austin, Austin, TX 78712
Corresponding Author: kajohnson{at}mail.utexas.edu
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 mM and the average rate of polymerization is 37 s-1, defining a specificity constant kcat/Km = 4.6 x 107 M-1s-1. Mismatched nucleotides show weaker ground-state nucleotide binding affinities ranging from 57-364 mM and slower rates of polymerization ranging from 0.013-1.16 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 to a T:T mismatch. Correctly base paired DNA dissociates from the polymerase at a rate of 0.02 s-1 promoting 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.
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