FUNCTIONAL AND MECHANISTIC STUDIES OF DNA TOPOISOMERASES

DNA 拓扑异构酶的功能和机制研究

• 批准号: 6179427
• 负责人: LEROY F LIU
• 金额: $ 35.04万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179427
• 关键词: DNA damage; DNA topoisomerases; Drosophilidae; Escherichia coli; Saccharomyces cerevisiae; alternatives to animals in research; apoptosis; bacterial genetics; enzyme mechanism; fungal genetics; genetic promoter element; hydrogen peroxide; molecular cloning; nucleic acid structure; oxidative stress; protein binding; protein isoforms; site directed mutagenesis; thiols; transcription factor; yeast two hybrid system

Our long-term objective is to understand the molecular mechanism and physiological function of multiple DNA topoisomerases. In the current application, we propose to study the roles of the two human topoisomerase II (TOP2) isoforms in chromosomal loop domain organization and apoptotic cell death. Many TOP2-targeted anti-cancer drugs are known to induce apoptotic cell death and high molecular weight (HMW) DNA fragmentation (about 50 kb). These HMW DNA fragments presumably reflect TOP2-mediated excision of chromosomal loop domains in which TOP2 is located at their loop anchorage sites. Strikingly, a similar pattern of HMW DNA fragmentation (predominantly 50 kb) has also been observed in apoptotic cells induced by diverse stimuli many of which are known to induce oxidative stress. The HMW DNA fragmentation has been suggested to be a committed step in the apoptotic cell death process, but the enzyme responsible for HMW DNA fragmentation has not been identified. Our preliminary studies have demonstrated that the two human DNA TOP2 isoforms, TOP2a and TOP2b, can be activated to become DNA cleaving "nucleases" by hydrogen peroxide, a reactive oxygen species (ROS) produced during oxidative stress. Oxidative stress has been suggested to be a potential cellular activation of TOP2b (and/or TOP2a) by hydrogen peroxide during oxidative stress is responsible for HMW DNA fragmentation in apoptotic cells. There are two major specific aims for the current application; (1). Functional studies of human TOP2 isoforms. Two approaches will be employed, identification of TOP2-interacting proteins and generating dominant negative mutant TOP2 cell lines. In addition to testing the roles of TOP2 isoforms in chromosomal loop domains mutated in patients with the Bloom's syndrome (genome instability) and WRN, mutated in patients with the Werner's syndrome (premature aging). (2). To establish the roles of human TOP2 isoforms in HMW DNA fragmentation during apoptotic cell death. We will determine if TOP2 ( and which TOP2 isoform) is activated into a "nuclease" in cells treated with hydrogen peroxide or other agents. We will also determine which TOP2 isoform is responsible for HMW DNA fragmentation during apoptotic cell death.

我们的长期目标是了解分子机制， 多种DNA拓扑异构酶的生理功能。在当前 应用，我们建议研究两个人类拓扑异构酶的作用， II（TOP2）亚型在染色体环结构域组织和凋亡中的作用 细胞死亡已知许多靶向TOP2的抗癌药物可诱导 凋亡性细胞死亡和高分子量（HMW）DNA片段化 （约50 kb）。这些HMW DNA片段可能反映了TOP 2介导的 切除其中TOP2位于它们的染色体环结构域， 线圈锚固部位。令人惊讶的是，高分子量DNA的相似模式 在细胞凋亡中也观察到片段化（主要是50 kb） 由多种刺激诱导的细胞，其中许多已知诱导 氧化应激高分子量DNA片段化被认为是一种 在凋亡细胞死亡过程中的一个重要步骤，但酶 负责HMW DNA片段化尚未确定。我们 初步研究表明，两个人类DNA TOP2 同种型，TOP 2a和TOP 2b，可以被激活，成为DNA切割 “核酸酶”是由过氧化氢，一种活性氧（ROS）产生 在氧化应激过程中。氧化应激被认为是一种 过氧化氢对TOP 2b（和/或TOP 2a）的潜在细胞激活 在氧化应激过程中， 凋亡细胞目前有两个主要的具体目标 应用;（1）.人TOP2同种型的功能研究。两 将采用方法鉴定TOP2相互作用蛋白 并产生显性负突变体TOP 2细胞系。除了 测试TOP2亚型在突变的染色体环结构域中的作用， 患有布鲁姆综合症（基因组不稳定）和WRN突变的患者 维尔纳综合征（过早衰老）的患者。（二）、到 建立人TOP2亚型在HMW DNA片段化过程中的作用 细胞凋亡我们将确定TOP2是否（以及TOP2亚型） 在用过氧化氢处理的细胞中被激活为“核酸酶”， 其他代理人。我们还将确定哪个TOP2亚型负责 凋亡细胞死亡过程中HMW DNA片段化。