Cytosine-5-methyltransferases add aldehydes to DNA.

Targeted methylation of cytosine residues by S-adenosylmethionine–dependent DNA methyltransferases modulates gene expression in vertebrates. Here we show that cytosine-5-methyltransferases catalyze reversible covalent addition of exogenous aliphatic aldehydes to their target residues in DNA, thus yielding corresponding 5-α-hydroxyalkylcytosines. Such atypical enzymatic reactions with non-cofactor-like substrates open new ways for sequence-specific derivatization of DNA and demonstrate enzymatic exchange of 5-hydroxymethyl groups on cytosine in support of an oxidative mechanism of DNA demethylation

5-Formylcytosine to Cytosine Conversion by C-C Bond Cleavage in vivo

Tet enzymes oxidise 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate to dC in the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. The question whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unsolved problem. Here we report the incorporation of synthetic isotope- and (R)-2’-fluorine-labelled dC and fdC-derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labelled fdC probe is efficiently converted into the corresponding labelled dC, most likely after its incorporation into the genome. This allows concluding that fdC is undergoing C-C bond cleavage in stem cells that leads to the direct re-installation of unmodified dC