Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase

J Biol Chem. 2001 Oct 12;276(41):38090-6. doi: 10.1074/jbc.M106045200. Epub 2001 Jul 26.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Pair Mismatch
  • Base Sequence
  • Biopolymers
  • DNA Primers
  • DNA-Directed DNA Polymerase / metabolism*
  • Humans
  • Kinetics
  • Mitochondria / enzymology*
  • Nucleotides / metabolism*
  • Protein Binding
  • Templates, Genetic


  • Biopolymers
  • DNA Primers
  • Nucleotides
  • DNA-Directed DNA Polymerase