title: Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase.
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 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.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas, Austin, 78712, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Johnson
authors.forename: K A
authors.initials: KA
authors.lastname: Johnson
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D001704:Biopolymers; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D013698:Templates, Genetic
chemical_list: D001704:Biopolymers; D017931:DNA Primers; D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase