SELECTIVITY OF DNA--CYTOSINE-5-METHYLTRANSERASE

此应用程序的最终目标是获取详细的 对这一机制的生理意义的理解 人DNA(胞嘧啶-5)甲基转移酶的作用 (E.C.2.1.1.37)建议的研究是由我们的 初步研究结果显示，这种高纯度的酶是 由于合成双链中存在错对而受到刺激 寡聚脱氧核苷酸。甲基化速率的提高 当含有30个双链时，获得了近15倍的倍数 将位于中心位置的d(PCG)位点的错配对与 不含错对的同源30个聚合体。几种类型的 错配已被证明有效地刺激了 酶的活性。碱基对不匹配的原因可能是 某些类型的DNA损伤，复制错误，形成 复合中间体和环状结构的形成 在DNA中。建议进行实验以研究作用模式。 DNA甲基转移酶的寡脱氧核苷酸模型 这些结构中的每一个。这种酶在这些情况下的行为 衬底结构将与关于 酶机制。对所提出的酶机制的测试将 包括使用活性部位定向的酶作用抑制物 基于含有寡聚脱氧核苷酸的5-氟胞苷。这个 抑制剂及其甲基膦酸类似物将用于 研究培养细胞中的甲基化。

The ultimate goal of this application is to obtain a detailed understanding of the physiological implications of the mechanism of action of the human DNA(cytosine-5)methyltransferase (E.C.2.1.1.37). The proposed studies are prompted by our preliminary findings showing that the highly purified enzyme is stimulated by the presence of mispairs in synthetic duplex oligodeoxynucleotides. An enhancement of methylation rate of nearly 15 fold has been obtained when duplex 30mers containing mispairs at a centrally located d(pCG) site were compared with homologous 30mers containing no mispairs. Several types of mismatch have been shown to be effective in stimulating the activity of the enzyme. Mismatched base pairs may result from certain types of DNA damage, replication errors, the formation of recombination intermediates, and the formation of loop structures in DNA. Experiments are proposed for studies of the mode of action of the DNA methyltransferase using oligodeoxynucleotide models of each of these structures. The behavior of the enzyme with these substrate structures will be correlated with proposals for the enzyme mechanism. Tests of the proposed enzyme mechanism will include the use of active site directed inhibitors of enzyme action based on 5-flourocytidine containing oligodeoxynucleotides. The inhibitors and their methylphosphonate analogs will be used to study methylation in cultured cells.