DNA TOPOISOMERASE II - DOMAINS, BINDING SITES & FUNCTION

该提议涉及来自酵母的DNA拓扑异构酶II。所述酶是 对细胞活力至关重要，而其人类对应物越来越多地 是抗肿瘤治疗重要靶点。研究酵母酶 提供了研究酶的结构和催化作用的机会。 详细的机制，由于材料的数量（数十 毫克）。此外，该基因的突变拷贝可以容易地被 重新引入到酵母细胞中，并分析与它们的相互作用。 原生细胞环境 这种酶很大（165 kD），催化一种复杂的反应--通道 DNA链通过DNA骨架中的酶桥接缺口。的 蛋白质的结构将被简化为更小的结构域， 反应机理将通过反应中间体的表征来定义。 初步结果将提供概念和实验框架 进行详细的分析。 将通过蛋白水解鉴定蛋白质的结构域。域 组织和酶活性将在有限的蛋白水解后测量 以测试多肽连续性的需要。重建 从合适的克隆片段中提取的活性酶将允许详细的 序列要求的测试，并可能解释ATP的要求 水解单晶将由完整的蛋白质和/或 域，以允许高分辨率结构测定。DNA 结合位点将通过一种新的方法绘制， 将蛋白质与DNA交联的能力以及对肽进行测序的能力 与DNA结合。反应中间体的表征将提供 对酶机制的现有模型进行严格测试。地区 DNA松弛所需的蛋白质将通过缺失来定义 TOP2基因分析拓扑异构酶II与其他 蛋白质将通过亲和层析和蛋白质 交联。将测试蛋白质的其他体内功能 通过替换突变的TOP2拷贝。 所描述的体外和体内研究将促进我们对以下方面的认识： 酵母拓扑异构酶II显着。这些信息将直接 应用于理解人类酶的功能，以及 用抑制性药物破坏这些功能的重要性。

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.