MUTATIONAL ANALYSIS OF E COLI DNA TOPOISOMERASE III

Dna拓扑异构酶是近年来广泛研究的主题。 几年来，特别是考虑到许多临床相关的 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 拓扑异构酶是该酶在体外的首选反应， 似乎是交织在一起的染色体的分离，而不是 染色体DNA超螺旋密度的调节。这个 本提案中详细介绍的实验旨在实现两个目标 具体目标：提供对TOPO III在 DNA代谢，并提供有关功能的信息 多肽的组织。利用一种新的诱变和选择方法 程序，建议分离和鉴定，在体外和 在体内，消除或改变细胞活性的突变 拓扑异构酶。具体地说，已提议： 1)获取一系列错误、无稽之谈、温度敏感(Ts) Topo III多肽内的突变。 2)突变型Topo III多肽的纯化及体外分析 酶的性质。 3)将突变组织到特定的类别中，这些类别在 拓扑异构酶催化的反应的不同方面 确定酶的结构域(即DNA结合)的努力 结构域、链通道结构域)。 4)将Topo III(TopB)编码基因的野生型副本替换为 特定类别突变的突变基因(包括 TopB的温度敏感等位基因)。 5)尝试关联突变体的体外生化特性 在多肽中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.