STRUCTURE-FUNCTION ANALYSIS OF MAMMALIAN DNA METHYLASE

哺乳动物 DNA 甲基化酶的结构功能分析

• 批准号: 3305747
• 负责人: NORBERT O. REICH
• 金额: $ 14.02万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-25 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305747
• 关键词: DNA; DNA binding protein; DNA footprinting; DNA methylation; active sites; chemical binding; chemical kinetics; crosslink; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; gel electrophoresis; high performance liquid chromatography; mass spectrometry; methyltransferase; molecular site; nucleic acid sequence; nucleic acid structure; protein sequence; synthetic nucleic acid

DNA methylation is one of numerous mechanisms whereby mammalian gene expression is regulated, and changes in DNA methylation are implicated in mammalian cellular transformation. Methylation patterns are established during gametogenesis and early embryogenesis through the action of DNA (cytosine-5-)-methyltransferase (DNA Mtase); these patterns are maintained by DNA Mtase, enabling the clonal propagation of patterns of gene expression during differentiation. Little is known about what determines the methylation patterns, and in particular, the involvement of DNA Mtase in this process. The recent availability of homogeneous Mtase preparations and the protein sequence provide the opportunity for detailed biochemical investigation of this important enzyme. Using homogeneous DNA Mtase isolated from Friend murine erythroleukemia cells and synthetic DNA substrates we propose to elucidate various aspects of Mtase-DNA interactions. Based on our experience with EcoRI DNA Mtase, we propose to develop a sequence-specific DNA binding assay. This will be used to map the enzyme-substrate interface with DNA- footprinting methods, thus providing detailed information about the size of the interface and whether major and/or minor grooves are contacted. Further, this will allow investigation of what role(s) the large (1000 amino acids) N-terminal domain plays in DNA binding and discrimination of hemi-methylated substrates. Regions of the enzyme involved in DNA and AdoMet recognition will be identified using several cross-linking strategies in combination with mass spectrometric analysis. The binding assay will be used to quantitate enzyme interactions with native, hemi-methylated and single stranded substrates. Comparison of these binding affinities with the corresponding specificity constants (k(cat)/K(m)) should aid our understanding of the enzyme's well known preference for hemi-methylated substrates. We will determine if the recently reported substrate inhibition occurs as a result of complex enzyme-enzyme interactions or through multiple DNA substrates binding the same enzyme molecule. The reported inhibition of enzyme activity deriving from "nonsubstrate nucleic acids" will be mechanistically investigated because of the possible regulatory importance. Results from the proposed experiments will be used in future studies of sequence- specificity and isolation of cellular factors which modulate Mtase activity.

DNA甲基化是哺乳动物基因的众多机制之一 表达受到调节，并且涉及 DNA 甲基化的变化 在哺乳动物细胞转化中。 甲基化模式是 在配子发生和早期胚胎发生过程中通过 DNA (胞嘧啶-5-)-甲基转移酶 (DNA Mtase) 的作用；这些图案 由 DNA Mtase 维持，从而实现模式的克隆传播 分化过程中的基因表达。 人们对什么知之甚少 决定甲基化模式，特别是参与 DNA Mtase 在此过程中的作用。 最近同质化的可用性 Mtase 制剂和蛋白质序列提供了机会 对这种重要酶的详细生化研究。 使用从 Friend 鼠红白血病中分离出的均质 DNA 转移酶 我们建议用细胞和合成 DNA 底物来阐明各种 Mtase-DNA 相互作用的各个方面。 根据我们在 EcoRI 方面的经验 DNA Mtase，我们建议开发一种序列特异性 DNA 结合测定。 这将用于绘制 DNA 酶-底物界面图 足迹法，从而提供有关尺寸的详细信息 界面的形状以及是否接触大凹槽和/或小凹槽。 此外，这将允许调查大型（1000 氨基酸）N 端结构域在 DNA 结合和区分中发挥作用 半甲基化底物。 参与 DNA 的酶区域 AdoMet 识别将使用多个交联来识别 结合质谱分析的策略。 结合测定将用于定量酶与 天然、半甲基化和单链底物。 比较 这些结合亲和力与相应的特异性常数 (k(cat)/K(m)) 应该有助于我们理解酶的众所周知的 对半甲基化底物的偏好。我们将确定是否 最近报道的底物抑制是由于复杂的结果而发生的 酶-酶相互作用或通过多种 DNA 底物结合 相同的酶分子。 报道的酶活性抑制 从“非底物核酸”衍生的将机械地 由于可能的监管重要性而进行调查。 结果来自 所提出的实验将用于未来的序列研究 调节 Mtase 的细胞因子的特异性和分离 活动。