Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase.

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