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和 多个DNA拓扑异构酶的生理功能。在电流中 应用，我们建议研究两个人拓扑酶的作用 II（TOP2）在染色体环域组织和凋亡中的同工型 细胞死亡。已知许多靶向2个靶向的抗癌药物会诱导 凋亡细胞死亡和高分子量（HMW）DNA碎片 （约50 kb）。这些HMW DNA片段大概反映了Top2介导的 TOP2所在的染色体环域的切除 循环锚地站点。令人惊讶的是，类似的HMW DNA模式 在凋亡中也观察到了碎裂（主要是50 kb） 由多种刺激诱导的细胞，其中许多已知会诱导 氧化应激。 HMW DNA碎片已被认为是 在凋亡细胞死亡过程中提出了一步，但是酶 尚未确定负责HMW DNA碎片。我们的 初步研究表明，两个人DNA TOP2 同工型TOP2A和TOP2B可以激活以变为DNA裂解 产生的活性氧（ROS）的过氧化氢的“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 在氧化应激期间是HMW DNA碎片的原因 凋亡细胞。当前有两个主要的特定目标 应用; （1）。人类TOP2同工型的功能研究。二 将采用方法，识别Top2相互作用蛋白 并产生显性的负突变型TOP2细胞系。此外 测试TOP2同工型在突变中突变的染色体环域中的作用 Bloom综合征（基因组不稳定性）和WRN的患者突变 患有Werner综合征的患者（过早衰老）。 （2）。到 在HMW DNA片段化中确定人类TOP2同工型的作用 凋亡细胞死亡。我们将确定top2（以及哪个top2同工型）是否 在用过氧化氢处理或 其他代理。我们还将确定哪种top2同工型负责 凋亡细胞死亡期间的HMW DNA片段化。