THREE-DIMENSIONAL STRUCTURE OF HHAI METHYLASE

HHAI 甲基化酶的三维结构

• 批准号: 2186817
• 负责人: Xiaodong Cheng
• 金额: $ 19.05万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186817
• 关键词: DNA; DNA binding protein; DNA methylation; S adenosylmethionine; SDS polyacrylamide gel electrophoresis; X ray crystallography; cofactor; crystallization; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; flucytosine; gel filtration chromatography; genomic imprinting; isomorphous substitution; methyltransferase; mutant

DNA methyltransferases are found in organisms ranging from bacteriophages to mammals. The function of methylases include protection of DNA against restriction enzymes as well as DNA mismatch repair in prokaryotes, and the control of gene expression, epigenesis, and genomic imprinting in eukaryotes. The long-term goal of this proposal is to understand from a structural standpoint how DNA-(cytosine-5)-methylases work with special emphasis on the mechanism of protein-DNA interactions. One such enzyme, M.HhaI methylase will be the focus of this study. The crystals of M.HhaI are the first useful crystals of a 5-cytosine methyltransferase. The three-dimensional structure(s) of M.HhaI in the presence of its cofactor AdoMet, the complex of M.HhaI with DNA, the covalent complex of M.HhaI with an oligonucleotide containing 5- fluorocytosine, and the complex between DNA and a mutant M.HhaI that binds to DNA tightly but is unable to methylate will be determined by the methods of X-ray crystallography. The structural information will enable the understanding of how M.HhaI recognizes a specific DNA sequence, and how it catalyses the methylation reaction. Due to the conserved nature of cytosine methylases, the implications of our knowledge on M.HhaI can be generalized to the entire family including the mammalian CpG methyltransferase. More significantly, the structural knowledge of specific DNA binding may enable us to design new methylases with altered specificities, which would be extremely useful tools for molecular biology and possibly for the human genome project.

DNA甲基转移酶存在于各种生物体中，从噬菌体到 对哺乳动物来说。甲基酶的功能包括保护DNA 抗限制性内切酶和DNA错配修复 原核生物与基因表达、表观发生和基因组的控制 真核生物的印记。这项提议的长期目标是 从结构角度理解DNA-(胞嘧啶-5)-甲基酶 特别强调蛋白质-DNA相互作用的机制。 一种这样的酶，海氏杆菌甲基酶将是这项研究的重点。这个 海氏支原体的晶体是5-胞嘧啶的第一个有用的晶体 甲基转移酶。《红楼梦》中海明威的三维结构(S) 它的辅因子ADOMet的存在，HhaI与DNA的络合物， HhaI与含5-脱氧核糖核酸的共价复合体 氟胞嘧啶，以及DNA与突变的Hhai之间的复合体 与DNA紧密结合但无法甲基化将由 X射线结晶学方法。结构信息将使 对M.HhaI如何识别特定DNA序列的理解，以及 它如何催化甲基化反应。由于自然界的保守 胞嘧啶甲基酶，我们的知识对M.HhaI的影响可以 推广到包括哺乳动物CpG在内的整个家族 甲基转移酶。更重要的是，结构知识 特异的DNA结合可能使我们能够设计出具有改变的甲基酶 特异性，这将是分子研究的非常有用的工具 生物学，可能还包括人类基因组计划。