Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine

A key step in the predicted mechanism of enzymatic transfer of methyl group s from 5-adenosyl-L-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C 5-MTase). Crystallographic analysis of complexes formed by HhaI methyltrans ferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5- fluorocytosine confirmed the existence of this dihydrocytosine intermediate . Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine a nalog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines i n C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonuc leotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylat ion by M.HhaI. Oligodeoxyribonudeotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhib itors of DNA methylation than oligodeoxyribonucleotides identical in sequen ce containing 5-fluorocytosine. Crystallographic analysis of a ternary comp lex involving M.HhaI, 5-adenosyl-L-homocysteine and a double-stranded 13-me r oligodeoxyribonucleotide containing DZCyt at the target position showed t hat the analog is flipped out of the DNA helix in the same manner as cytosi ne, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a cova lent bond was detected between the sulfur atom of the catalytic site nucleo phile, cysteine 81, and the pyrimidine C6 carbon. These results indicate th at DZCyt can occupy the active site of M.HhaI as a transition state mimic a nd, because of the high degree of affinity of its interaction with the enzy me, it can act as a potent inhibitor of methylation. (C) 1999 Academic Pres s.