DNA BINDING BY HUMAN TOPOISOMERASE I

这项研究的总体目标是验证一系列假说，这些假说与人类拓扑异构酶I与DNA的相互作用有关，这些假说是基于最近解开的酶-DNA复合体的晶体结构。拓扑异构酶I的晶体结构表明，蛋白质中必须有铰链点，才能使酶在释放和重新结合DNA时以钳子的形式打开和关闭。这些铰链点的位置将使用定点突变和生化分析相结合的方法来确定，并将尝试在没有DNA的情况下确定蛋白质的结构。定点突变将被用来评估连接封闭构象中两个相反环的盐桥的重要性。当被锁定在封闭构型中时，酶可以沿着DNA滑动的程度将被确定。建议进行实验，以测试蛋白质在DNA结合时的二聚化，或类似于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的优先结合提供基础。蛋白质的连接区形成一个从酶伸出的很长的螺旋状延伸部分，具有未知的功能。使用交联技术，将评估连接物与溶液中结合的DNA的接近程度。改变连接体中选定的碱性氨基酸对松弛反应的影响也将被确定。这一假设，即连接子在细胞核中介导蛋白质-蛋白质相互作用，可能涉及更高级别的结构，将使用缺乏连接子的缺失变体来检验。人类拓扑异构酶I的一系列衍生物将通过突变技术产生，这些突变技术改变蛋白质的帽区，以确定该区域在超螺旋DNA松弛过程中通常是阻碍还是促进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.