STRUCTURE-FUNCTION ANALYSIS OF MAMMALIAN DNA METHYLASE

DNA甲基化是哺乳动物基因 表达受到调控，并牵涉到DNA甲基化的变化 在哺乳动物的细胞转化中。甲基化模式有 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；这些模式 由DNA MTase维持，使模式的克隆繁殖成为可能 分化过程中基因表达的变化。人们对什么知之甚少 决定甲基化模式，特别是，参与 DNA MTase在这一过程中的作用。同类产品的最新供应情况 MTase制剂和蛋白质序列提供了机会 对这种重要的酶进行详细的生化研究。 从Friend小鼠红白血病中分离的同源DNA MTase 细胞和合成DNA底物，我们建议阐明各种 MTase-DNA相互作用的几个方面。根据我们在EcoRI方面的经验 DNA MTase，我们建议建立一种序列特异性DNA结合分析方法。 这将被用来绘制酶-底物与DNA的界面- 足迹方法，从而提供有关大小的详细信息 以及是否接触主要和/或次要凹槽。 此外，这将允许调查(S)扮演什么角色的大(1000 氨基酸)N-末端结构域在DNA结合和识别中的作用 半甲基化的底物。涉及DNA和DNA的酶的区域和 将使用几个交叉链接来标识ADOMet识别 策略与质谱分析相结合。 结合试验将被用来定量酶与 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众所周知的 偏爱半甲基化底物。我们将确定是否 最近报道的底物抑制是由于络合物 酶-酶相互作用或通过多个DNA底物结合 同样的酶分子。已报道的酶活性抑制 从“非底物核酸”衍生出来的将是机械的 由于可能的监管重要性而进行了调查。结果来自 拟议的实验将用于未来对序列的研究- 调节MTase的细胞因子的特异性和分离 活动。

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.