On the mechanism of demethylation of 5-methylcytosine in DNA

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–2H3]-5-methylcytosine in DNA. A mechanism of the DNA demethylation reaction is proposed based on this observation.