title: The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by 5-azacytosine is likely to involve methyl transfer to the inhibitor.
abstract: The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by the mechanism-based inhibitor 5-azacytosine has remained unclear, mainly because of the unavailability of a substrate in which the inhibitor, but not normal cytosine, is present at the target site. We synthesized an oligonucleotide duplex containing a single target site for the EcoRII methyltransferase, in which the target base is 5-azacytosine. This substrate formed a stable covalent complex with EcoRII methyltransferase in the absence and in the presence of the cofactor S-adenosylmethionine. The complex formed in the presence of the cofactor was resistant to SDS and moderate heat treatment, and a methyl group was incorporated into the complex. Enzyme titration and kinetic studies of inhibition suggest that methyl transfer to the complex occurred only during the first turnover of the reaction. These results suggest that, when the enzyme binds to 5-azacytosine in the presence of the cofactor, a methyl group is transferred to the N-5 position of the base, resulting in the inactivation of the enzyme.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gabbara
authors.forename: A S
authors.initials: AS
authors.lastname: Bhagwat
mesh_terms: D001374:Azacitidine / Q000494:pharmacology*; D001483:Base Sequence; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D015257:DNA-Cytosine Methylases / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D008745:Methylation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
chemical_list: D009841:Oligonucleotides; D012967:Sodium Dodecyl Sulfate; D012436:S-Adenosylmethionine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine