DNA TOPOISOMERASE II--DOMAINS, BINDING SITES & FUNCTIONS

这项提案涉及酵母中的DNA拓扑异构酶II。这种酶是 对细胞存活至关重要，而它的人类同行是越来越多的 是抗肿瘤治疗的重要靶点。酵母酶的研究 为研究酶的结构和催化作用提供了机会 详细的机制，由于可用材料的数量(数万 毫克)。此外，基因的突变拷贝很容易被 重新导入酵母细胞，并分析其与其相互作用 原生蜂窝环境。 该酶很大(165kD)，并催化一个复杂的反应-通道 DNA链通过DNA主干上的酶桥接缝隙。这个 蛋白质的结构将被还原为更小的结构域，而催化 反应机理将通过反应中间产物的表征来确定。 初步结果将提供概念和实验框架 以便进行详细的后续分析。 蛋白质的结构域将通过蛋白质分解来确定。域 在有限的蛋白质降解后，将测量组织和酶的活性 测试多肽连续性的要求。重组 来自适当克隆片段的活性酶将允许详细说明 顺序要求的测试，并可能解释对ATP的要求 水解液。将由完整的蛋白质和/或 其结构域，以允许高分辨率结构测定。脱氧核糖核酸 将通过一种新的方法绘制结合位点图，该方法结合了 能够将蛋白质与DNA交联，并能够对多肽进行排序 绑定到DNA上。对反应中间体的表征将提供 对酶作用机制的现有模型进行了严格的测试。地区 DNA松弛所需的蛋白质将通过缺失来定义 TOP2基因的分析。拓扑异构酶II与其他 蛋白质将通过亲和层析和蛋白质鉴定 交联性。将测试该蛋白质的其他体内功能。 通过与TOP2的突变拷贝移位。 所描述的体外和体内研究将促进我们对 酵母菌拓扑异构酶II显著。这一信息将直接 应用于理解人类酶的功能，以及 使用抑制性药物扰乱这些功能的重要性。

This proposal concerns DNA topoisomerase II from yeast. The enzyme is essential to cell viability, and its human counterpart is an increasingly important target for antitumor therapeutics. Studying the yeast enzyme offers the opportunity to investigate the enzyme's structure and catalytic mechanism in detail, due to the quantities of material available (tens of milligrams). Furthermore, mutated copies of the gene can readily be reintroduced into yeast cells and analyzed for interactions with their native cellular milieu. The enzyme is large (165 kD) and catalyzes a complex reaction- the passage of DNA strands through an enzyme-bridged gap in the DNA backbone. The protein's structure will be reduced to smaller domains and the catalytic mechanism will be defined by characterization of reaction intermediates. The initial results will provide the conceptual and experimental framework for a subsequent analysis in great detail. Structural domains of the protein will be identified by proteolysis. Domain organization and enzyme activity will be measured after limited proteolysis to test the requirement for polypeptide continuity. Reconstitution of active enzyme from appropriate cloned fragments will permit detailed testing of sequence requirements and may explain the requirement for ATP hydrolysis. Single crystals will be grown of the intact protein and/or domains thereof, to permit a high resolution structure determination. DNA binding sites will be mapped by a new methodology which combines the ability to cross-link protein to DNA with the ability to sequence peptides bound to DNA. Characterization of a reaction intermediate will provide stringent tests of current models for the enzyme's mechanism. Regions of the protein required for DNA relaxation will be defined by deletion analysis of the TOP2 gene. Contacts between topoisomerase II and other proteins will be identified by affinity chromatography and protein cross-linking. Additional in vivo functions of the protein will be tested by transplacement with mutated copies of TOP2. The in vitro and in vivo studies described will advance our knowledge of yeast topoisomerase II significantly. This information will have direct application to an understanding of the functions of the human enzyme, and of the importance of disrupting such functions with inhibitory drugs.