The ultimate goal of this application is to obtain a detailed understanding of the physiological implications of the mechanism of action of the human DNA(cytosine-5)methyltransferase (E.C.2.1.1.37). The proposed studies are prompted by our preliminary findings showing that the highly purified enzyme is stimulated by the presence of mispairs in synthetic duplex oligodeoxynucleotides. An enhancement of methylation rate of nearly 15 fold has been obtained when duplex 30mers containing mispairs at a centrally located d(pCG) site were compared with homologous 30mers containing no mispairs. Several types of mismatch have been shown to be effective in stimulating the activity of the enzyme. Mismatched base pairs may result from certain types of DNA damage, replication errors, the formation of recombination intermediates, and the formation of loop structures in DNA. Experiments are proposed for studies of the mode of action of the DNA methyltransferase using oligodeoxynucleotide models of each of these structures. The behavior of the enzyme with these substrate structures will be correlated with proposals for the enzyme mechanism. Tests of the proposed enzyme mechanism will include the use of active site directed inhibitors of enzyme action based on 5-flourocytidine containing oligodeoxynucleotides. The inhibitors and their methylphosphonate analogs will be used to study methylation in cultured cells.
