2‐Pyrimidinone as a probe for studying the Eco RII DNA methyltransferase–substrate interaction

Eco RII DNA methyltransferase (M.EcoRII) recognizes the 5′…CC*T/AGG…3′ DNA sequence and catalyzes the transfer of the methyl group from S ‐adenosyl‐ l ‐methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2‐pyrimidinone, a cytosine analogue lacking an NH 2 group at the C4 position of the pyrimidine ring. Also, DNA containing 2‐pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2‐Pyrimidinone was incorporated into the 5′…CCT/AGG…3′ sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2‐pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2‐pyrimidinone and to a greater destabilizing influence of 2‐pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2‐pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the Eco RII recognition sequence is not involved in the DNA–M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2‐Pyrimidinone replacing the target cytosine in the EcoR II recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S ‐adenosyl‐ l ‐methionine, proton transfer to the C5 position of 2‐pyrimidinone occurs and in the presence of S ‐adenosyl‐ l ‐methionine, methyl transfer to the C5 position of 2‐pyrimidinone occurs.