DNA CYTOSINE C5 METHYLTRANSFERASE

DNA甲基化对正常哺乳动物发育至关重要， 抑制负责DNA甲基化的酶[DNA胞嘧啶 C（5）甲基转移酶，DCMTase]有助于减轻肿瘤发生。 的 哺乳动物DCMT酶拟议表征的长期目标 是开发具有潜在应用的新型抑制剂， 抗癌药 1)第一个具体目标是确定 哺乳动物DCMTase的胞嘧啶甲基化机制和 结构特征的细菌DCMT酶，M.Hhal. 目标是 提供事件的详细动力学描述， 底物添加，并以甲基转移步骤结束。 这 信息对于评价抑制剂效力和 对抑制剂作用的机械理解。 该动力学分析 也将成为定量分配影响的基础， 命名为DCMTase突变体。 这一具体目标将通过 预稳态动力学方法，分配分析，稳态和预稳态 稳态荧光光谱和活性位点诱变 残基 2)第二个具体目标是描述 用单链DNA观察到哺乳动物DCMT酶抑制。 的DNA 对这种有效抑制作用至关重要的结构特征将是 鉴定 假设大的，N-末端结构域的 将检测DCMTase与抑制剂的结合。 3)的 第三个具体目标是确定哺乳动物DCMTse是否可以催化 5-甲基胞嘧啶脱氨生成胸腺嘧啶。 其他人已经 提出这种致突变反应可以解释许多人类遗传学原因， 包括癌症在内的疾病。 然而，人们的反应只是 用细菌DCMTases证明。

DNA methylation is essential for normal mammalian development, and inhibition of the enzyme responsible for DNA methylation [DNA cytosine C(5) methyltransferase, DCMTase] aids in alleviating oncogenesis. The long-term goal of the proposed characterization of the mammalian DCMTase is the development of novel inhibitors with potential applications as anticancer drugs. 1) The first specific aim is to determine the mechanism of cytosine methylation by the mammalian DCMTase and the structurally characterized bacterial DCMTase, M.Hhal. The goal is to provide a detailed kinetic description of the events starting with substrate addition, and ending with the methyl transfer step. This information is essential for the evaluation of inhibitor potency and for a mechanistic understanding of inhibitor action. This kinetic analysis will also form the basis for a quantitative assignment of the effects of designated DCMTase mutants. This specific aim will be addressed using pre-steady state kinetic methods, partition analysis, steady-and pre- steady state fluorescence spectroscopy, and mutagenesis of active site residues. 2) The second specific aim is to characterize the mechanism of mammalian DCMTase inhibition observed with single-stranded DNA. The DNA structural features that are essential for this potent inhibition will be identified. The hypothesis that the large, N-terminal domain of the DCMTase, is involved in binding the inhibitor will be tested. 3) The third specific aim is to determine if the mammalian DCMTse can catalyze the deamination of 5-methylcytosine to generate thymine. Others have proposed that this mutagenic reaction may account for many human genetic diseases including cancer. However, the reaction has only been demonstrated with bacterial DCMTases.