SELECTIVITY OF DNA--CYTOSINE-5-METHYLTRANSERASE

此应用程序的最终目标是获得详细的 了解该机制的生理含义 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 初步研究结果表明，高度纯化的酶是 由于合成双链体中存在错配而刺激 寡脱氧核苷酸 甲基化率的提高， 当双链体30聚体含有 将位于中心的d（pCG）位点的错配与 同源30聚体，不含错配。 几种类型的 错配已被证明是有效的刺激， 酶的活性。 不匹配的碱基对可能是由 某些类型的DNA损伤，复制错误， 重组中间体，以及环结构的形成 在DNA中。 提出了实验研究的作用方式 的DNA甲基转移酶使用寡脱氧核苷酸模型， 每一个这些结构。 酶的行为与这些 基板结构将与建议， 酶机制 对所提出的酶机制的测试将 包括使用酶作用的活性位点定向抑制剂 基于含5-氟胞苷的寡脱氧核苷酸。 的 抑制剂及其甲基膦酸酯类似物将用于 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.