Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5methyltransferase ofE.coliK-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine

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Abstract
The product of the dcm gene Is the only DNA cytosine C5 methyltransferase of Escherlchla coli K-12; it catalyses transfer of a methyl group from Sadenosyl methlonine (SAM) to the C-5 position of the inner cytosine residue of the cognate sequence CCATGG. Sequence-specific, covalent crosslinking of the enzyme to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine is demonstrated. This reaction is abolished if serine replaces the cysteine at residue 177 of the enzyme. These results lend strong support to a catalytic mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine residue for electrophllic attack by the methyl donor SAM. The enzyme is capable of self-methylatlon in a DNA-independent reaction requiring SAM and the presence of cysteine at position 177.