FUNCTIONAL AND MECHANISTIC STUDIES OF DNA TOPOISOMERASES

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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.