The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition 
of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. 
Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification 
that can alter gene activity and chromosomal stability, influencing both 
differentiation and tumorigenesis. Thus, it is important to understand the 
critical mechanistic determinants of ZCyt's inhibitory action. Although several 
DNA C5-MTases have been reported to undergo essentially irreversible binding to 
ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl 
transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate 
that formation of stable complexes between HhaI methyltransferase (M.HhaI) and 
oligodeoxyribonucleotides containing ZCyt at the target position for methylation 
(ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and 
AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing 
conditions and take on a compact conformation that increases their 
electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can 
occur only in the presence of AdoMet, these results suggest (1) that the 
inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, 
tightly closed conformation of M.HhaI that prevents DNA and co-factor release 
and (2) that methylation of ZCyt in DNA is not required for inhibition of 
M.HhaI.