Fidelity of nucleotide insertion at 8-oxo-7,8-dihydroguanine by mammalian DNA polymerase delta. Steady-state and pre-steady-state kinetic analysis

J Biol Chem. 2001 Feb 9;276(6):3764-71. doi: 10.1074/jbc.M006696200. Epub 2000 Nov 10.


Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K(m)). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Animals
  • Base Sequence
  • Cattle
  • Cytidine Triphosphate / metabolism*
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • DNA Primers
  • Guanine / analogs & derivatives*
  • Guanine / metabolism*
  • Humans
  • Kinetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Recombinant Proteins / metabolism


  • DNA Primers
  • Proliferating Cell Nuclear Antigen
  • Recombinant Proteins
  • 8-hydroxyguanine
  • Guanine
  • Cytidine Triphosphate
  • Adenosine Triphosphate
  • DNA Polymerase III