ROLE OF TOPOISOMERASES IN DNA METABOLISM

本项目的目标是为生物化学提供基础。 拓扑异构酶在DNA代谢中的作用。这些酶显然是 与DNA复制过程中DNA链的管理有关， 转录和重组。在原核生物中，拓扑异构酶是 临床上有意义的抗生素的靶标，而在真核细胞中，它们 是很有前途的抗肿瘤药物的靶点。因此，一个完整的 对他们的行动模式的理解应该为 合理的药物治疗。拓扑异构酶功能的研究 用纯化的重组系统模拟它们的体外作用 蛋白质。 拓扑异构酶作用的一个中心点是在 DNA复制，当DNA合成必须停止时，女儿 染色体必须解开。使用小质粒DNA 进行单向或双向复制，我们将研究 三种大肠埃希菌拓扑异构酶、DNA旋转酶、 拓扑异构酶I(Topo I)和拓扑异构酶III(Topo III) DNA复制的终止和分离阶段。性情 在复制分叉终止后，将确定分叉组件 以及Tus蛋白催化的细胞停滞的作用机制 复制分叉进展被阐明。这种蛋白质，当络合到 特定的终止子序列，导致在E. Coli染色体DNA复制。更完整的决赛画面 复制过程的各个阶段应提供以下有用信息 我们对分类所需事件的不断发展的理解 染色体和细胞分裂的启动。 将研究Topo III在转录过程中的可能作用 通过探索该实验室最近的一项观察，这一点 酶能与RNA形成共价复合体。Topo III的能力 对RNA起催化作用(即，断裂并重新密封链)将是 确定和开发的分析方法证明Topo III催化 RNA的拓扑调控。 Topo III还可以抑制酵母和大肠杆菌中的重组 (来自该实验室以及其他实验室的初步数据)。似乎 很可能这种酶的作用是破坏某些重组的 中级的。这种重组抑制的生物化学基础 将通过研究Topo III对各种连接的影响来进行 DNA分子和RecA催化的链转移反应 重组体系中的缺失形成和同源配对 同时进行体外复制(以提供单个 启动重组所需的链)和重组。 希望在这一批款期结束时，有一个更全面的情况 将会出现关于拓扑异构酶控制的DNA和 RNA链，特别是允许它的Topo III的性质 参与DNA新陈代谢的三个主要方面：复制， 转录和重组。

It is the goal of this project to provide a biochemical basis for the role of topoisomerases in DNA metabolism. These enzymes are clearly implicated in the management of DNA strands during DNA replication, transcription, and recombination. In prokaryotes, topoisomerases are the target of clinically significant antibiotics, while in eukaryotes they are the target of promising antineoplastic agents. Thus a full understanding of their mode of action should provide a sounder basis for rational drug therapy. Topoisomerase function is investigated by modeling their action in vitro using systems reconstituted with purified proteins. One central point of topoisomerase action is during the final stages of DNA replication, when DNA synthesis must cease and the daughter chromosomes must be untangled. Using systems where small plasmid DNAs undergo either unidirectional or bidirectional replication, we will study the influence of the three Escherichia coli topoisomerases, DNA gyrase, topoisomerase I (Topo I), and topoisomerase III (Topo III) on the termination and segregation stages of DNA replication. The disposition after termination of the replication fork components will be determined and the mechanism of action of Tus protein-catalyzed arrest of replication fork progression elucidated. This protein, when complexed to specific terminator sequences, causes replication termination during E. coli chromosomal DNA replication. A more complete picture of the final stages of the replication process should provide useful information for our developing understanding of the events necessary for the sorting of chromosomes and the initiation of cell division. The possible action of Topo III during transcription will be investigated by exploring fully a recent observation from this laboratory that this enzyme can form a covalent complex with RNA. The ability of Topo III to act catalytically on RNA (i. e., break and reseal strands) will be determined and assays developed to demonstrate Topo III-catalyzed topological modulation of RNA. Topo III can also act to suppress recombination in yeast and E. coli (preliminary data from this laboratory as well as others). It seems likely that the enzyme acts to destabilize some recombinational intermediate. The biochemical basis of this recombination suppression will be approached by examining the effect of Topo III on various joined DNA molecules and RecA-catalyzed strand transfer reactions, as well as on deletion formation and homologous pairing in reconstituted systems in vitro simultaneously undergoing replication (to provide the single strands required for the initiation of recombination) and recombination. It is hoped that at the end of this grant period a more complete picture will have emerged concerning topoisomerase managed trafficking of DNA and RNA strands and in particular of the properties of Topo III that allow it to participate in three major aspects of DNA metabolism: replication, transcription, and recombination.