All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It 
has been proposed that this cysteine initiates catalysis by attacking the C6 of 
cytosine and thereby activating the normally inert C5 position. We show here 
that substitutions of this cysteine in the E. coli methylase M. EcoRII with 
either serine or tryptophan results in a complete loss of ability to transfer 
methyl groups to DNA. Interestingly, mutants with either serine or glycine 
substitution bind tightly to substrate DNA. These mutants resemble the wild-type 
enzyme in that their binding to substrate is not eliminated by the presence of 
non-specific DNA in the reaction, it is sensitive to methylation status of the 
substrate and is stimulated by an analog of the methyl donor. Hence the 
conserved cysteine is not essential for the specific stable binding of the 
enzyme to its substrate. However, substitution of the cysteine with the bulkier 
tryptophan does reduce DNA binding. We also report here a novel procedure for 
the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA 
substrate for M. EcoRII in which the target cytosine is replaced by 
5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms 
an irreversible complex with the enzyme. As expected, this modified substrate 
does not form irreversible complexes with the mutants.