ROLE OF TOPOISOMERASES IN DNA METABOLISM

该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 涉及DNA复制过程中DNA链的管理， 转录和重组。 在原核生物中，拓扑异构酶是 靶向的临床重要抗生素，而在真核生物中， 都是有前途的反间谍特工的目标 因此， 了解他们的行动方式应该提供一个更健全的基础， 合理的药物治疗 拓扑异构酶功能的研究， 使用纯化的重组系统在体外模拟它们的作用 proteins. 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， 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 三种大肠杆菌拓扑异构酶，DNA促旋酶， 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。 处置 在终止复制后，将确定分叉组件 以及Tus蛋白催化的细胞凋亡抑制的作用机制 复制叉进展阐明。 当这种蛋白质与 特定的终止子序列，导致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有用的信息， 我们对事件的理解， 染色体和细胞分裂的起始。 拓扑异构酶III在转录过程中的可能作用将被研究 通过充分探索这个实验室最近的观察， 酶可以与RNA形成共价复合物。 Topo III的能力， 对RNA起催化作用（i.即，断裂和重新密封股）将被 确定并开发试验以证明Topo III催化的 RNA的拓扑调控 拓扑异构酶III还可以抑制酵母和大肠杆菌中的重组。杆菌 （来自该实验室以及其他实验室的初步数据）。 似乎 这种酶的作用可能是破坏某些重组蛋白的稳定性， 中间体 这种重组抑制的生化基础 将通过研究拓扑三的影响，对各种加入 DNA分子和RecA催化的链转移反应，以及 重组系统中的缺失形成和同源配对 体外同时进行复制（以提供单个 起始重组所需的链）和重组。 希望在本赠款期结束时， 将出现关于拓扑异构酶管理的DNA贩运， RNA链，特别是拓扑异构酶III的特性， 参与DNA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.