Open AccessSequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5methyltransferase ofE.coliK-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine
Author(s) -
Theodor Hanck,
Sabine Schmidt,
HansJoachim Fritz
Publication year - 1993
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/21.2.303
Subject(s) - cytosine , oligonucleotide , cysteine , dna , residue (chemistry) , biology , enzyme , methyltransferase , serine , biochemistry , stereochemistry , microbiology and biotechnology , methylation , chemistry
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.