FUNCTIONAL AND MECHANISTIC STUDIES OF DNA TOPOISOMERASES

我们的长期目标是了解分子机制和 多种DNA拓扑异构酶的生理功能。在当前 应用，我们建议研究这两种人类拓扑异构酶的作用 染色体环区组织中的II(TOP2)亚型与细胞凋亡 细胞死亡。已知许多TOP2靶向抗癌药物可诱导 细胞凋亡性死亡与高分子量DNA片段化 (约50kb)。这些HMW DNA片段可能反映了TOP2介导的 TOP2位于其上的染色体环域的切除 环状锚地。令人惊讶的是，类似的HMW DNA模式 在细胞凋亡中也观察到片段化(主要是50kb)。 由不同刺激诱导的细胞，其中许多已知诱导 氧化应激。HMW DNA的断裂被认为是一种 在细胞凋亡过程中迈出的重要一步，但这种酶 对HMW DNA碎裂负有责任的人尚未确定。我们的 初步研究表明，这两种人类DNA TOP2 TOP2a和TOP2b亚型可以被激活，成为DNA裂解 过氧化氢产生的一种活性氧物种(ROS)产生的“核酸酶” 在氧化应激过程中。氧化应激被认为是一种 过氧化氢对TOP2b(和/或TOP2A)的潜在细胞激活作用 在氧化应激过程中导致HMW DNA断裂 凋亡性细胞。当前有两个主要的具体目标 申请；(1)。人类TOP2亚型的功能研究。二 将采用鉴定TOP2相互作用蛋白的方法 产生显性负性突变型TOP2细胞系。除了……之外 检测TOP2亚型在中国人染色体环区突变中的作用 患有Bloom综合征(基因组不稳定)和WRN的患者发生突变 在沃纳综合征(早衰)患者中。(2)。至 建立人TOP2亚型在HMW DNA断裂中的作用 细胞凋亡性死亡我们将确定TOP2(以及TOP2的哪个亚型) 在用过氧化氢或过氧化氢处理的细胞中被激活为“核酸酶” 其他特工。我们还将确定哪种TOP2亚型负责 HMW DNA在凋亡细胞死亡过程中的断裂。

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.