MUTATIONAL ANALYSIS OF E COLI DNA TOPOISOMERASE III

DNA拓扑异构酶是近年来研究的热点 多年来，特别是考虑到许多临床相关的事实， 抗生素和抗肿瘤药物专门针对这些酶。 大肠杆菌DNA拓扑异构酶III（Topo III）是一种新的 拓扑异构酶是酶的优选反应，在体外， 似乎是相互缠绕的染色体的分离，而不是 染色体DNA超螺旋密度的调节。 的 本建议中详细介绍的实验旨在实现两个 具体目标：了解Topo III在以下方面的作用： DNA代谢，并提供有关功能的信息 多肽的组织。 使用一种新的诱变和选择 程序，建议在体外和 在体内，消除或改变所述细胞的活性的突变可导致细胞内的细胞凋亡。 拓扑异构酶 具体而言，建议： 1)获得一系列错义、无义和温度敏感（ts）的 Topo III多肽内的突变。 2)纯化突变体Topo III多肽并分析体外 酶的性质。 3)将突变组织成特定的类别， 不同方面的反应催化的拓扑异构酶在一个 努力定义酶的结构域（即，DNA结合 结构域，链通道结构域）。 4)将编码Topo III（topB）的基因的野生型拷贝替换为 特定类型突变的突变基因（包括 topB的温度敏感等位基因）。 5)尝试将突变体的体外生化特性与 在多肽内具有特定结构变化的酶。

DNA topoisomerases have been the subject of extensive research in recent years, particularly in view of the fact that many clinically relevant antibiotics and antitumor drugs specifically target these enzymes. Escherichia coli DNA topoisomerase III (Topo III) is a novel topoisomerase in that the preferred reaction of the enzyme, in vitro, appears to be the separation of intertwined chromosomes rather than the modulation of the superhelical density of chromosomal DNA. The experiments detailed in this proposal are designed to achieve two specific goals: to provide an understanding of the role of Topo III in DNA metabolism, and to provide information as to the functional organization of polypeptide. Using a novel mutagenesis and selection procedure, it is proposed to isolate and characterize, both in vitro and in vivo, mutations that eliminate or alter the activity of the topoisomerase. Specifically, it has been proposed to: 1) obtain a series of missense, nonsense, and temperature sensitive (ts) mutations within the Topo III polypeptide. 2) purify the mutant Topo III polypeptides and analyze the in vitro properties of enzymes. 3) organize the mutations into specific classes that are defective in different aspects of the reactions catalyzed by topoisomerases in an effort to define structural domains of the enzyme (i.e., DNA binding domain, strand passage domain). 4) replace the wild type copy of the gene encoding Topo III (topB) with mutant genes of the specific classes of mutations (including a temperature sensitive allele of topB). 5) attempt to correlate the in vitro biochemical properties of the mutant enzymes with specific structural changes within the polypeptide.