Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E. coli K-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine.
The product of the dcm gene is the only DNA cytosine-C5 methyltransferase of Escherichia coli K-12; it catalyses transfer of a methyl group from S-adenosyl methionine (SAM) to the C-5 position of the inner cytosine residue of the cognate sequence CCA/TGG. 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 electrophilic attack by the methyl donor SAM. The enzyme is capable of self-methylation in a DNA-independent reaction requiring SAM and the presence of cysteine at position #177.
Hanck
T
T
Institut für Molekulare Genetik, Georg-August-Universität Göttingen, Germany.
Schmidt
S
S
Fritz
H J
HJ
D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*
D003902:Detergents; D009838:Oligodeoxyribonucleotides; D003841:Deoxycytidine; D003596:Cytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; C007746:5-fluoro-2'-deoxycytidine