Highly purified DNA methyltransferase from human placenta methylates hemimethylated and 5-methylcytosine-free DNA substrates suggesting that one enzyme molecule may exercise both, the maintenance and de novo activities. Modification of these methyl accepting polymers with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, anti (BPDE) interferes with the methylation reaction, and this inhibition is proportional to the degree of BPDE-modification. This indicates that BPDE - DNA adducts affect both the maintenance and the de novo DNA methyltransferase activities. The mechanism responsible for such inhibition is related neither to interference of BPDE - DNA adducts with the initial binding of the enzyme to DNA nor with the processive mode of action of the enzyme on the modified DNA template. More likely, the BPDE - DNA adducts inhibit the transmethylation reaction directly at the sites of modification.
Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.
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