DNA CYTOSINE C5 METHYLTRANSFERASE

DNA 胞嘧啶 C5 甲基转移酶

• 批准号: 2734836
• 负责人: NORBERT O. REICH
• 金额: $ 13.4万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734836
• 关键词: 5 methylcytosine; DNA binding protein; DNA methylation; animal tissue; enzyme activity; enzyme inhibitors; enzyme substrate; fluorescence microscopy; methyltransferase; nucleic acid structure; single strand conformation polymorphism; site directed mutagenesis

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.

DNA甲基化是哺乳动物正常发育所必需的，而且 抑制负责DNA甲基化的酶[DNA胞嘧啶 C(5)甲基转移酶，DCMTase]有助于减轻肿瘤的发生。这个 哺乳动物DCMTase特性研究的长期目标 是开发具有潜在应用的新型缓蚀剂 抗癌药物。1)第一个具体目标是确定 哺乳动物DCMTase胞嘧啶甲基化的机制 细菌DCMTase的结构特征，M.Hhal。我们的目标是 提供事件的详细动态描述，从 底物加入，并以甲基转移步骤结束。这 信息对于评价缓蚀剂的效力和 从机理上理解抑制剂的作用。这一动力学分析 也将成为量化分配影响的基础 指定的DCMTase突变体。这一具体目标将通过以下方式实现 稳态前动力学方法、配分分析、稳态和稳态前 稳态荧光光谱与活性部位的诱变 残留物。2)第二个具体目标是描述 用单链DNA观察到哺乳动物的DCMTase抑制。DNA 对这种强有力的抑制至关重要的结构特征将是 确认身份。假设大的，N-末端的结构域 DCMTase，参与结合的抑制物将被检测。3) 第三个特定目的是确定哺乳动物的DCMTse是否能够催化 5-甲基胞嘧啶脱氨生成胸腺嘧啶。其他人则有 提出了这种突变反应可能解释了许多人类基因 包括癌症在内的疾病。然而，人们的反应只是 用细菌DCMTase证明。