Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine.

Abstract

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.5-Methylcytosine, a minor base in the DNA of a variety of organisms, is formed by postreplicative methylation of DNA by S-adenosylmethionine (AdoMet) in reactions catalyzed by DNA-cytosine methyltransferases (DCMTases). In recent years, much evidence has been obtained which indicates that 5-methylcytosine residues in DNA play an important role in eukaryotic gene expression (for reviews see refs. 1 and 2). Consequently, there has been wide interest in the pyrimidine analog 5-azacytidine (azaCyd), which inhibits formation of 5-methylcytosine in DNA and results in dramatic effects on gene expression and cell differentiation (e.g., see refs. 3-9).Current evidence indicates that the mechanism by which azaCyd causes decreased DNA methylation involves incorporation of 5-azacytosine (azaC) into DNA and subsequent inhibition of DCMTase. Incorporation of small amounts of azaC into DNA of mammalian cells results in a loss of DCMTase activity in extracts obtained from such cells (5,10). Further, incubation of DNA containing azaC (azaC-DNA) with mammalian or bacterial DCMTases results in a very potent inhibition of enzyme activity (11, 12), but kinetic studies have not revealed the mechanism of inhibition. On the basis of the probable catalytic mechanism of DCMTases and known chemical properties of azaC, we recently speculated that the mechanism o...

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