DNA BINDING BY HUMAN TOPOISOMERASE I

拟议研究的总体目标是测试一系列关于人类拓扑异构酶I与DNA相互作用的假设，这些假设基于最近解决的酶-DNA复合物的晶体结构。拓扑异构体I的晶体结构表明，蛋白质内部一定存在铰链点，以允许酶在释放和重新结合DNA时像夹子一样打开和关闭。这些铰点的位置将使用位点定向诱变和生化分析的组合来确定，并将尝试确定缺乏DNA的蛋白质结构。位点定向诱变将用于评估在封闭构象中连接两个相反环的盐桥的重要性。当酶被锁定在封闭结构中时，它能沿着DNA滑动的程度将被确定。研究人员提出了实验来测试蛋白质在DNA结合时的二聚化，或者类似同源结构域的第二个DNA结合位点的存在是否为酶与超螺旋DNA的优先结合提供了基础。蛋白质的连接区域形成一个非常长的盘绕状延伸，从酶中突出，具有未知的功能。使用交联技术，将评估连接体与溶液中结合DNA的接近程度。改变连接剂内选定的碱性氨基酸对弛豫反应的影响也将被确定。连接子介导细胞核中可能涉及高阶结构的蛋白质-蛋白质相互作用的假设将通过缺乏连接子的缺失变体进行验证。人类拓扑异构酶I的一系列衍生物将通过诱变技术产生，这些技术改变蛋白质的帽区，以确定该区域在超螺旋DNA松弛期间是否通常阻碍或促进DNA旋转。

The overall goal of the proposed research is to test a series of hypotheses which are concerned with the interaction of human topoisomerase I with DNA and which are based on the recently-solved crystal structure of enzyme-DNA complexes. The crystal structure of topoisomerese I suggests that there must be hinge points within the protein to allow the enzyme to open and close as a clamp as it releases and rebinds DNA. The location of these hinge points will be determined using a combination of site-directed mutagenesis and biochemical assays, and attempts will be made to determine the structure of the protein in the absence of DNA. Site-directed mutagenesis will be used to assess the importance of a salt bridge that connects the two opposing loops in the closed conformation. The extent to which the enzyme can slide along the DNA when locked in the closed configuration will be determined. Experiments are proposed to test whether dimerization of the protein upon DNA binding, or the presence of a second DNA binding site resembling homeodomains provide the basis for the preferential binding of the enzyme to supercoiled DNA. The linker region of the protein forms a very long coiled-coil extension that protrudes from the enzyme and has an unknown function. Using crosslinking techniques, the proximity of the linker to the bound DNA in solution will be evaluated. The effects of changing selected basic amino acids within the linker on the relaxation reaction will also be determined. The hypothesis that the linker mediates protein-protein interactions in the nucleus possibly involving higher order structures will be tested using a deletion variant lacking the linker. A series of derivatives of human topoisomerase I will be generated by mutagenesis techniques that alter the cap region of the protein to determine whether this region normally hinders or facilitates DNA rotation during the relaxation of supercoiled DNA.