title: On the mechanism of demethylation of 5-methylcytosine in DNA.
abstract: DNA methylation is an important biological process that programmes gene expression in vertebrates. The methylation pattern is generated by a combination of methylation and demethylation reactions catalyzed by DNA methyltransferases and putative demethylases. MBD2 binds methylated DNA and possesses DNA demethylase activity. We use here direct analysis of the reaction mixture by GC-MS using a water-tolerant gas chromatographic column to avoid the loss of potential volatile products and identify the leaving residue of the demethylation reaction. We show that the DNA demethylase reaction catalyzed by a recombinant human MBD2 purified from SF9 insect cells releases dideuteroformaldehyde from [Me-(2)H(3)]-5-methylcytosine in DNA. A mechanism of the DNA demethylation reaction is proposed based on this observation.
authors.affiliation: Department of Chemistry, McGill University, Montreal, QB, Canada.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hamm
authors.forename: George
authors.initials: G
authors.lastname: Just
authors.forename: Nathalie
authors.initials: N
authors.lastname: Lacoste
authors.forename: Nicolas
authors.initials: N
authors.lastname: Moitessier
authors.forename: Moshe
authors.initials: M
authors.lastname: Szyf
authors.forename: Orval
authors.initials: O
authors.lastname: Mamer
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D000818:Animals; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D008958:Models, Molecular
chemical_list: D004268:DNA-Binding Proteins; C489325:MBD2 protein, human; D044503:5-Methylcytosine; D004247:DNA; D015254:DNA Modification Methylases