Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site.
Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.
Prieto Alamo
M J
MJ
Unité 347 INSERM, 80 Rue du Général Leclerc, 94276 Le Kremlin Bicêtre Cedex, France.
Jurado
J
J
Francastel
E
E
Laval
F
F
D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli