The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by the 
mechanism-based inhibitor 5-azacytosine has remained unclear, mainly because of 
the unavailability of a substrate in which the inhibitor, but not normal 
cytosine, is present at the target site. We synthesized an oligonucleotide 
duplex containing a single target site for the EcoRII methyltransferase, in 
which the target base is 5-azacytosine. This substrate formed a stable covalent 
complex with EcoRII methyltransferase in the absence and in the presence of the 
cofactor S-adenosylmethionine. The complex formed in the presence of the 
cofactor was resistant to SDS and moderate heat treatment, and a methyl group 
was incorporated into the complex. Enzyme titration and kinetic studies of 
inhibition suggest that methyl transfer to the complex occurred only during the 
first turnover of the reaction. These results suggest that, when the enzyme 
binds to 5-azacytosine in the presence of the cofactor, a methyl group is 
transferred to the N-5 position of the base, resulting in the inactivation of 
the enzyme.