Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine.
DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.
D001374:Azacitidine; D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004262:DNA Restriction Enzymes; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases
D004247:DNA; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; D001374:Azacitidine