title: A common mechanism of action for the N-glycosylase activity of DNA N-glycosylase/AP lyases from E. coli and T4.
abstract: Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. These N-glycosylases are known to have associated AP lyase activities. In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites. Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities. Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates. For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)). Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively. Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.
authors.affiliation: Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Purmal
authors.forename: L E
authors.initials: LE
authors.lastname: Rabow
authors.forename: G W
authors.initials: GW
authors.lastname: Lampman
authors.forename: R P
authors.initials: RP
authors.lastname: Cunningham
authors.forename: Y W
authors.initials: YW
authors.lastname: Kow
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D045647:DNA Glycosylases; D004260:DNA Repair / Q000502:physiology*; D043382:DNA-Formamidopyrimidine Glycosylase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006898:Hydroxylamines / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D014508:Urea / Q000378:metabolism; D014764:Viral Proteins
chemical_list: D029968:Escherichia coli Proteins; D006898:Hydroxylamines; D014764:Viral Proteins; C017064:benzyloxyamine; D014508:Urea; C005214:methoxyamine; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli