TOPOISOMERASES AND DNA REPLICATION

拓扑异构酶和 DNA 复制

• 批准号: 2177497
• 负责人: KENNETH J MARIANS
• 金额: $ 25.87万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177497
• 关键词: TOPOISOMERASES; DNA; REPLICATION

Topoisomerases are ubiquitous enzymes that alter the linking number of DNA. As such, they are likely to be involved in all aspects of DNA metabolism. Their importance is underscored by the fact that in eukaryotes they are the target of potent anti-cancer agents, whereas in prokaryotes, both DNA gyrase and topoisomerase IV (Topo IV), which was purified and characterized during the previous grant period, are targets of the most potent broad-spectrum antibiotics known (e.g., ciprofloxacin). Thus, it is crucial to understand their mechanism of action and the roles they play during DNA metabolism. During the previous grant period, using pBR322 and oriC DNA replication reconstituted with purified proteins, we have shown that the dour E. coli topoisomerases, Topo I, Topo II (DNA gyrase), Topo III, and Topo IV are distinct in their ability to support the various stages of theta-type DNA replication. During the new grant period, we will investigate the biological, biochemical, and structural basis for the differences in topoisomerase action. We have shown that Topos II-IV can all support nascent chain elongation in vitro. Evidence indicating that gyrase is the only enzyme involved in this step in vivo is equivocal. To determine the contributions of Topos III and IV in supporting this step in vivo, we will construct strains carrying various combinations of mutations in these enzymes and determine the effect on DNA synthesis. We have shown that Topo III, a type I topoisomerase that requires a single-stranded DNA-binding site, supports nascent chain elongation. This suggests, because no single-strands exist ahead of the forks, that Topo III acts behind the forks. We also know that both Topos III and IV act more efficiently at this stage if they are present during initiation, suggesting that a preferential binding site is formed at the origin. We will determine whether such binding sites exist and investigate how the excess positive windings formed during theta-type DNA replication are distributed over the template and how this distribution affects the action of the topoisomerases. Topo IV and gyrase show high homology, yet during replication only Topo IV, not gyrase, can support processing of the late intermediate and decatenation of the daughter molecules. On the other hand, only gyrase, not Tops IV, and supercoil DNA. We will investigate the structural basis for these differences by constructing chimeric Topo IV-gyrase molecules and assessing the associated activities and by solving the crystal structure of Topo IV. Topo IV acts subsequent to termination of replication and prior to partition. Thus, it is likely to be involved in a signal transduction pathway that connects DNA replication and cell division. To detect other proximal gene products involved in this pathway, we will isolate high- copy suppressors of dominant-lethal Topo IV mutations.

拓扑异构酶是无处不在的酶，改变了链接数的链接数 脱氧核糖核酸。 因此，它们可能参与DNA的各个方面 代谢。 在 真核生物是有效抗癌代理的目标，而在 原核生物，DNA回旋酶和拓扑异构酶IV（topo IV），这是 纯化和表征在上一个赠款期间是目标 已知的最有效的广谱抗生素（例如， 环丙沙星）。 因此，了解他们的机制至关重要 动作及其在DNA代谢中扮演的角色。 在上一个赠款期间，使用PBR322和ORIC DNA复制 用纯化的蛋白质重构，我们已经表明了大肠杆菌 拓扑异构酶，Topo I，Topo II（DNA Gyrase），Topo III和Topo IV是 他们支持theta型DNA的各个阶段的能力很明显 复制。 在新的赠款期间，我们将调查 生物学，生化和结构基础的差异 拓扑异构酶作用。 我们已经表明，Topos II-IV都可以支持新生的链伸长 体外。 证据表明回旋酶是唯一涉及的酶 在此步骤中，体内是模棱两可的。 确定 Topos III和IV在体内支持此步骤时，我们将构建 这些酶中携带各种突变组合的菌株和 确定对DNA合成的影响。 我们已经证明了Topo III，这是I型topoisomerase，需要一个 单链DNA结合位点支持新生的链伸长。 这表明，因为在叉子之前没有单链，所以 Topo III在叉子后面行动。 我们也知道Topos III和IV都 如果在启动过程中存在，则在此阶段更有效地采取行动 表明优先结合位点是在原点形成的。 我们 将确定是否存在这种结合位点，并研究 在theta型DNA复制过程中形成的过量正绕组是 分布在模板上以及该分布如何影响 拓扑异构酶的作用。 Topo IV和回旋酶显示出高的同源性，但在复制过程中仅托普 iv而不是回旋酶，可以支持晚期中间体的处理 女儿分子的衰老。 另一方面，只有回旋酶， 不是顶部IV和超级旋转DNA。 我们将研究结构基础 对于这些差异，通过构建嵌合topo iv- gyrase分子 并评估相关活动并通过解决晶体 TOPO IV的结构。 TOPO IV在终止复制之后的行动，然后 分割。 因此，它可能参与信号转导 连接DNA复制和细胞分裂的途径。 检测其他 涉及该途径的近端基因产品，我们将分离高 - 主导致命topo IV突变的复制抑制器。