FUNCTIONAL AND MECHANISTIC STUDIES OF DNA TOPOISOMERASES

DNA 拓扑异构酶的功能和机制研究

• 批准号: 6018516
• 负责人: LEROY F LIU
• 金额: $ 29.13万
• 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/6018516
• 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并 多种DNA拓扑异构酶的生理功能。在当前 应用，我们建议研究两种人类拓扑异构酶的作用 染色体环结构域组织和细胞凋亡中的 II (TOP2) 同工型 细胞死亡。已知许多 TOP2 靶向抗癌药物会诱导 细胞凋亡和高分子量 (HMW) DNA 断裂 （约 50 kb）。这些 HMW DNA 片段可能反映了 TOP2 介导的 切除染色体环结构域，其中 TOP2 位于其 环锚固地点。引人注目的是，HMW DNA 具有相似的模式 在细胞凋亡中也观察到碎片（主要是 50 kb） 由多种刺激诱导的细胞，其中许多刺激已知会诱导 氧化应激。 HMW DNA 片段化被认为是 细胞凋亡过程中的关键步骤，但酶 造成 HMW DNA 断裂的原因尚未确定。我们的 初步研究表明，两种人类DNA TOP2 同种型 TOP2a 和 TOP2b 可被激活以进行 DNA 切割 过氧化氢（一种活性氧 (ROS)）产生的“核酸酶” 在氧化应激期间。氧化应激被认为是 过氧化氢对 TOP2b（和/或 TOP2a）的潜在细胞激活 氧化应激期间 HMW DNA 断裂是造成 凋亡细胞。当前有两个主要具体目标 应用; (1).人类 TOP2 亚型的功能研究。二 将采用方法，鉴定 TOP2 相互作用蛋白 并产生显性失活突变TOP2细胞系。此外 测试 TOP2 同工型在染色体环结构域中突变的作用 患有布鲁姆综合症（基因组不稳定）和 WRN 突变的患者 患有维尔纳综合症（过早衰老）的患者。 （2）。到 确定人类 TOP2 同种型在 HMW DNA 片段化过程中的作用 细胞凋亡。我们将确定是否是 TOP2（以及哪种 TOP2 同工型） 在用过氧化氢处理的细胞中被激活成“核酸酶”或 其他代理。我们还将确定哪种 TOP2 同工型负责 细胞凋亡过程中 HMW DNA 断裂。