DNA BINDING BY HUMAN TOPOISOMERASE I

拟议的研究的总体目标是测试一系列的假设，这些假设与人类拓扑异构酶I与DNA的相互作用有关，并且这些假设是基于最近解决的酶-DNA复合物的晶体结构。 拓扑异构酶I的晶体结构表明，蛋白质内必须有铰链点，以允许酶在释放和重新结合DNA时像夹子一样打开和关闭。 这些铰链点的位置将使用定点诱变和生物化学测定的组合来确定，并且将尝试在不存在DNA的情况下确定蛋白质的结构。将使用定点诱变来评估在闭合构象中连接两个相对环的盐桥的重要性。 将确定当酶锁定在闭合构型中时酶可沿着DNA滑动的程度。 提出实验来测试是否二聚化的蛋白质DNA结合后，或类似同源结构域的第二个DNA结合位点的存在下提供的基础上的优先结合的酶超螺旋DNA。 蛋白质的接头区域形成从酶突出的非常长的卷曲螺旋延伸，并且具有未知的功能。 使用交联技术，将评价接头与溶液中结合的DNA的接近度。还将确定改变接头内所选碱性氨基酸对弛豫反应的影响。 将使用缺乏接头的缺失变体来测试接头介导细胞核中可能涉及更高级结构的蛋白质-蛋白质相互作用的假设。 一系列的衍生物的人拓扑异构酶I将产生诱变技术，改变帽区的蛋白质，以确定该地区是否通常阻碍或促进DNA旋转过程中的超螺旋DNA的松弛。

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.