title: 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity.
abstract: Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodG.dA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG.dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.
authors.affiliation: Biology Division, National Cancer Center Research Institute, Tokyo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Tchou
authors.forename: H
authors.initials: H
authors.lastname: Kasai
authors.forename: S
authors.initials: S
authors.lastname: Shibutani
authors.forename: M H
authors.initials: MH
authors.lastname: Chung
authors.forename: J
authors.initials: J
authors.lastname: Laval
authors.forename: A P
authors.initials: AP
authors.lastname: Grollman
authors.forename: S
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D043382:DNA-Formamidopyrimidine Glycosylase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli