Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the 
reaction pathway, have been deployed extensively in the analysis of metabolic 
pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA 
methyltransferases (C5 MTases) by oligodeoxynucleotides containing 
5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a 
well-documented example of mechanism-based inhibition of enzymes central to 
nucleic acid metabolism. Here, we describe the interaction between the C5 MTase 
from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex 
containing 2-H pyrimidinone, an analogue often referred to as zebularine and 
known to give rise to high-affinity complexes with MTases. X-ray crystallography 
has demonstrated the formation of a covalent bond between M.HhaI and the 2-H 
pyrimidinone-containing oligodeoxynucleotide. This observation enables a 
comparison between the mechanisms of action of 2-H pyrimidinone with other 
mechanism-based inhibitors such as FdC. This novel complex provides a molecular 
explanation for the mechanism of action of the anti-cancer drug zebularine.