EcoRII DNA methyltransferase () recognizes the 5¢ CC*T/AGG 3¢ DNA sequence and catalyzes the transfer of the methyl group fromS-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). Here, we study themechanismof byDNA containing 2-pyrimidinone, a cytosine analogue lacking an NH2 group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts with functional groups of pyrimidine bases of the recognition sequence