MECHANISMS OF TOPOISOMERASE POISONS

拓扑异构酶中毒的机制

• 批准号: 6138715
• 负责人: HIROSHI HIASA
• 金额: $ 20.57万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138715
• 关键词: DNA damage; DNA gyrase; DNA replication; DNA replication origin; DNA topoisomerases; Escherichia coli; Staphylococcus aureus; cytotoxicity; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme reconstitution; enzyme substrate; enzyme substrate complex; helicase; molecular cloning; protein purification; quinoline; recombinant DNA; synthetic nucleotide

Topoisomerase are ubiquitous enzymes that alter the linking number of DNA. As such, they play essential roles in every aspect of DNA metabolism. Their importance is underscored by the fact that in eukaryotes these enzymes are the cellular targets of potent anticancer drugs, whereas in prokaryotes both DNA Gyrase and topoisomerase IV (Topo IV) are targets of the most potent broad-spectrum antibacterial agents (e.q., ciprofloxacin). These drugs convert topoisomerases to poisons that inhibit DNA replication and lead to double-strand break (DSB) generation. Thus, it is crucial to understand the molecular basis of the cytotoxicity of these topoisomerase inhibitors. I have shown that the encounter of a replication fork with a Topo IV- quinolone-DNA ternary complex converts the ternary complex to a nonreversible form. (I) We will examine if a complete replication fork is required for the conversion of a ternary complex to a nonreversible form. It is possible that the DnaB helicase alone is the active agent. (II) We will study the effects of a topoisomerase trapped at a site of DNA damage on the replication fork. It has been demonstrated that DNA lesions, such as an apurinic site, stimulate eukaryotic topoisomerase- mediated DNA cleavages. We will examine if an apurinic site also acts as a topoisomerase poison for bacterial enzymes. We will compare effects of an apurinic site-induced Topo IV-DNA complex and a Topo IV- quinolone-DNA ternary complex on the replication fork to determine if these two distinct mechanisms of poisoning topoisomerases have the same consequences. (III) Recent studies have demonstrated that quinolones do not stimulate S. aureus DNA gyrase (Gyrase)-mediated DNA cleavages. We will characterize S. aureus Gyrase and a ternary complex formed with this enzyme. Furthermore, we will examine if DNA strand cleavage is always required for replication fork arrest by a topoisomerase- quinolone-DNA ternary complex. (IV) We have proposed that quinolone- induced DSB generation is a two-step process. Using an assay where replication forks collide with a ternary complex, I detected an activity in an E. coli extract that could generate DSBs at dead-end topoisomerase complexes. We will identify the protein(s) required for the second step of DSB formation and complete the reconstitution of the DSB process in vitro with purified-proteins.

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，它们在DNA的各个方面都起着重要的作用 代谢。 在 真核生物这些酶是有效抗癌的细胞靶 药物，而在原核生物中，DNA陀螺酶和拓扑异构酶IV（TOPO） iv）是最有效的广谱抗菌剂的目标 （E.Q.，环丙沙星）。 这些药物将拓扑异构酶转化为毒药 抑制DNA复制并导致双链断裂（DSB） 一代。因此，了解理解的分子基础至关重要 这些拓扑异构酶抑制剂的细胞毒性。 我已经证明了复制叉的相遇带有topo iv- 喹诺酮-DNA三元络合物将三元络合物转换为 非可逆形式。 （i）我们将检查是否完全复制叉 将三元络合物转换为不可逆的需要 形式。 单独的DNAB解旋酶可能是活性剂。 （ii）我们将研究被困在地点的拓扑异构酶的影响 复制叉上的DNA损伤。 已经证明了DNA 病变，例如肾上腺位，刺激真核拓扑异构酶 - 介导的DNA裂解。 我们将检查一个载质网站是否也起作用 作为细菌酶的拓扑异构酶毒药。 我们将比较 源自位点诱导的TOPO IV-DNA复合物和topo iv-的影响 复制叉上的喹诺酮-DNA三元复合物，以确定是否是否 中毒拓扑异构酶的这两种不同的机制具有相同的 结果。 （iii）最近的研究表明喹诺酮类 不刺激金黄色葡萄球菌DNA回旋酶（Gyrase）介导的DNA裂解。 我们 将表征金黄色葡萄球菌和形成的三元复合物 这种酶。此外，我们将检查DNA链裂解是否为 拓扑异构酶 - 喹诺酮-DNA三元复合物。 （iv）我们提出了喹诺酮 - 诱导的DSB生成是一个两步的过程。 使用测定法 复制叉与三元复合物相撞，我检测到了一种活动 在大肠杆菌提取物中，该提取物可以在死端拓扑异构酶下生成DSB 复合物。 我们将确定第二步所需的蛋白质 DSB成立并完成DSB过程的重组 纯化蛋白质的体外。