Aberrant cytosine methylation in promoter regions leads to gene silencing 
associated with cancer progression. A number of DNA methyltransferase inhibitors 
are known to reactivate silenced genes; including 5-azacytidine and 
2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less 
cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic 
basis for this difference, we carried out a detailed comparisons of the 
interaction between purified DNA methyltransferases and 
oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 
2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When 
incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by 
both nucleosides is essentially identical. However, the stability and 
reversibility of the enzyme complex in the absence and presence of cofactor 
differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that 
are irreversible when the 5-azacytosine ring is intact. ODNs containing 
2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of 
both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the 
ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the 
cofactor S-adenosyl methionine are maintained through the formation of a 
reversible covalent interaction. The differing stability and reversibility of 
the covalent bonds may partially account for the observed differences in 
cytotoxicity between zebularine and 5-azacytidine inhibitors.