The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 
2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation 
in the human genome. Proposals for the enzyme's mechanism of action suggest that 
5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine 
intermediate. We have used an oligodeoxynucleotide containing 
5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the 
dihydrocytosine intermediate. Gel retardation experiments demonstrate the 
formation of the expected covalent complex between duplex DNA containing 
5-fluorodeoxycytidine and the human enzyme. Formation of the complex was 
dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting 
that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. 
Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination 
in aqueous solution. Because C-to-T transition mutations are especially 
prevalent at CG sites in human DNA, we have used high-performance liquid 
chromatography to search for thymidine that might be generated by hydrolysis 
during the methyl transfer reaction. Despite the potential for deamination 
inherent in the formation of the intermediate, the methyltransferase did not 
produce detectable amounts of thymidine. The data suggest that the ability of 
the human methyltransferase to preserve genetic information when copying a 
methylation pattern (i.e., its fidelity) is comparable to the ability of a 
mammalian DNA polymerase to preserve genetic information when copying a DNA 
sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA 
does not appear to be due to the normal enzymatic maintenance of methylation 
patterns.