MECHANISTIC ANALYSIS OF SITE DIRECTED MUTANT NUCLEASE ENZYMES

定点突变核酸酶的机理分析

• 批准号: 6470648
• 负责人: BINGHUI SHEN
• 金额: $ 12.12万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6470648
• 关键词: biomedical resource; genome; human tissue; technology /technique

The major objective of the proposed research is to understand the interrelationship between the structure and function of a newly identified nuclease human flap endonuclease-1 (FEN-1) in the context of DNA repair. Current biochemical and genetic data have demonstrated that FEN-1 is a structure specific endonuclease. It may play critical roles in DNA replication, repair, and recombination. However, sufficient evidence is not available for this class of nucleases to establish a structural basis for their unique biochemical and physiological functions and their structural and functional relationships. Our preliminary data on the analysis of biochemical domains has suggested that there are three functional domains in human FEN-1 protein responsible for the functions of DNA substrate binding, catalysis, protein-protein interaction, and nuclear localization. The proposed research is designed to establish the detailed structural understanding of the catalytic cen ter for DNA substrate recognition, binding and cleavage. and to analyze the structural elements responsible for protein-protein interaction and nuclear localization. We will examine functional alterations through site-directed mutagenisis of each functional domain. The mutant proteins will be characterized by our newly developed kinetic flow cytometry, biochemical assays and crystallographic structural analysis, and green fluorescence driven microscopy.

拟议研究的主要目标是了解 新结构与功能之间的相互关系 在上下文中鉴定出核酸酶人皮瓣核酸内切酶-1 (FEN-1) DNA 修复。 目前的生化和遗传数据表明 FEN-1是一种结构特异性核酸内切酶。 它可能发挥关键作用 DNA 复制、修复和重组中的作用。 然而， 没有足够的证据表明此类核酸酶 为其独特的生化和结构奠定基础 生理功能及其结构和功能 关系。 我们的生化分析初步数据 域表明人类存在三个功能域 FEN-1蛋白负责DNA底物结合的功能， 催化、蛋白质-蛋白质相互作用和核定位。 这 拟议的研究旨在建立详细的结构 了解 DNA 底物识别的催化中心， 结合和裂解。 并分析结构要素 负责蛋白质-蛋白质相互作用和核定位。 我们将通过现场定向检查功能变更 每个功能域的诱变。 突变蛋白将是 以我们新开发的动态流式细胞术为特征， 生化分析和晶体结构分析，以及绿色 荧光驱动显微镜。