MECHANISTIC ANALYSIS OF SITE DIRECTED MUTANT NUCLEASE ENZYMES

本研究的主要目的是了解 一个新的生物体的结构和功能之间的相互关系 鉴定的核酸酶人瓣核酸内切酶-1（FEN-1） DNA修复 目前的生物化学和遗传学数据表明 FEN-1是结构特异性内切核酸酶。 它可能会起到关键作用， 在DNA复制、修复和重组中的作用。 然而，在这方面， 没有足够的证据表明这类核酸酶可以 为它们独特的生物化学和 生理功能及其结构和功能 关系。 我们对生化分析的初步数据 结构域的研究表明，在人类中存在三个功能结构域， 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。 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。 并分析其结构元素 负责蛋白质-蛋白质相互作用和核定位。 我们将通过现场指导检查功能改变 每个功能结构域的诱变。 突变蛋白质将 通过我们新开发的动态流式细胞术表征， 生化分析和晶体结构分析，以及绿色 荧光驱动显微镜。

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.