Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase.


We have examined the fidelity of polymerization catalyzed by the human ...
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 microm, and the average rate of polymerization is 37 x s(-1), defining a specificity constant kcat/Km = 4.6 x 10(7) x m(-1) x s(-1). Mismatched nucleotides show weaker ground-state nucleotide binding affinities ranging from 57 to 364 microm and slower rates of polymerization ranging from 0.013 to 1.16 x 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 x 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.



Kinetic Parameters, Fidelity, Nucleotide Incorporation


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