COVALENT BOND FORMATION BETWEEN A DNA-CYTOSINE METHYLTRANSFERASE AND DNA CONTAINING 5-AZACYTOSINE
COVALENT BOND FORMATION BETWEEN A DNA-CYTOSINE METHYLTRANSFERASE AND DNA CONTAINING 5-AZACYTOSINE
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DOI：
10.1073/pnas.81.22.6993
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发表时间：
1984-01-01
期刊：
影响因子：
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通讯作者：
GARRETT, CE
中科院分区：
文献类型：
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作者：
SANTI, DV;NORMENT, A;GARRETT, CE
含有5-氮胞嘧啶(AzaC)的DNA是一种有效的DNA胞嘧啶甲基转移酶抑制剂。AzaC-DNA与Hpa II甲基酶形成共价复合体，Hpa II甲基酶是一种将C-C-G-C序列的内部C甲基化的细菌酶。该络合物在至少3天内不会发生可检测到的解离，并且对十二烷基硫酸钠变性是稳定的。在用DNase和磷酸二酯酶广泛消化该复合体后，凝胶过滤得到与.apprx结合的甲基酶。1相当于氮杂C；消化后的复合体具有与天然酶相似的表观分子量。虽然Hpa II内切酶处理azaC-DNA对甲基酶的结合影响很小，但用同样切割C-C-G-G序列的Msp I内切酶处理后，结合作用显著减少，这表明Hpa II识别序列中的azaC残基是甲基酶共价相互作用的重要组成部分。然而，由于存在残留结合，DNA中其他地方的azaC残基也可能与甲基酶共价结合。这些结果解释了为什么azaC-DNA是胞嘧啶甲基转移酶的如此有效的抑制剂，以及如何将如此低水平的azaC掺入DNA中可以导致胞嘧啶甲基化的显著减少。最后，考虑胞嘧啶甲基酶的可能催化机制和氮杂碳的化学性质表明，抑制作用至少部分是活性中心定向的过程，并允许对共价络合物的结构提出建议。
DNA [Escherichia coli] containing 5-azacytosine (azaC) is a potent inhibitor of DNA-cytosine methyltransferases. AzaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-C sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with sodium dodecyl sulfate. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to .apprx. 1 equivalent of azaC; the digested complex had an apparent MW 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 sequences 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 methylase 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.