title: Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein, a 1-methyladenine-DNA dioxygenase.
abstract: The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of alpha-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5'-phosphorylated 1-me-dAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5' phosphate group was required. Consequently, 1-me-dAMP(5') was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-me-dATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-me-dATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts.
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
authors.forename: Pertti
authors.initials: P
authors.lastname: Koivisto
authors.forename: Tod
authors.initials: T
authors.lastname: Duncan
authors.forename: Tomas
authors.initials: T
authors.lastname: Lindahl
authors.forename: Barbara
authors.initials: B
authors.lastname: Sedgwick
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D001667:Binding, Competitive; D018736:DNA Adducts / Q000378:metabolism; D019175:DNA Methylation; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D018736:DNA Adducts; D029968:Escherichia coli Proteins; D009711:Nucleotides; D009841:Oligonucleotides; D011061:Poly A; C015465:poly(dA); C008407:1-methyladenine; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli; C499236:ALKBH1 protein, human; D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D004256:DNA Polymerase I; D045643:DNA Repair Enzymes; D000225:Adenine