Enhanced chemical stability of adomet analogues for improved methyltransferase-directed labeling of DNA.

Methyltransferases catalyze specific transfers of methyl groups from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet) to various nucleophilic positions in biopolymers like DNA, RNA, and proteins. We had previously described synthesis and application of AdoMet analogues carrying sulfonium-bound 4-substituted but-2-ynyl side chains for transfer by methyltransferases. Although useful in certain applications, these cofactor analogues exhibited short lifetimes in physiological buffers. Examination of the reaction kinetics and products showed that their fast inactivation followed a different pathway than observed for AdoMet and rather involved a pH-dependent addition of a water molecule to the side chain. This side reaction was eradicated by synthesis of a series of cofactor analogues in which the separation between an electronegative group and the triple bond was increased from one to three carbon units. The designed hex-2-ynyl moiety-based cofactor analogues with terminal amino, azide, or alkyne groups showed a markedly improved enzymatic transalkylation activity and proved well suitable for methyltransferase-directed sequence-specific labeling of DNA in vitro and in bacterial cell lysates

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