THREE-DIMENSIONAL STRUCTURE OF HHAI METHYLASE

HHAI 甲基化酶的三维结构

• 批准号: 2186818
• 负责人: Xiaodong Cheng
• 金额: $ 20万
• 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/2186818
• 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甲基转移酶存在于从噬菌体到其他生物体中