8-oxodGTP incorporation by DNA polymerase beta is modified by active-site residue Asn279.
To understand how the active site of a DNA polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA polymerase beta (pol beta) and two active-site mutants. We compared the coding of these polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol beta, there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5'-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol beta; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation.
Miller
H
H
Department of Pharmacological Sciences, SUNY Stony Brook, Stony Brook, New York 11794-8651, USA. miller@pharm.sunysb.edu
Prasad
R
R
Wilson
S H
SH
Johnson
F
F
Grollman
A P
AP
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000235:genetics / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009153:Mutagens / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
D003848:Deoxyguanine Nucleotides; D009153:Mutagens; D009838:Oligodeoxyribonucleotides; C078206:8-oxodeoxyguanosine triphosphate; D001216:Asparagine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019951:DNA Polymerase beta; D003849:Deoxyguanosine