Fidelity of nucleotide insertion at 8-oxo-7,8-dihydroguanine by mammalian DNA polymerase delta. Steady-state and pre-steady-state kinetic analysis.
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.
Einolf
H J
HJ
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Guengerich
F P
FP
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003570:Cytidine Triphosphate / Q000378:metabolism*; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III