The proposed project aims at the elucidation of molecular mechanisms of anticancer drugs targeting DNA topoisomerases and the physiological functions of mammalian DNA topoisomerases II beta, III alpha and III beta. The potential roles of mammalian DNA topoisomerases IIbeta, IIIalpha and III beta in cancer chemotherapy will also be assessed. A two prong experimental approach will be taken. For the well- characterized enzymes, a combination of biochemical, genetic, and X-ray crystallographic studies will be done to achieve a detailed understanding of drug-enzyme-DNA interactions. These studies will focus on drugs that act by either inhibiting eukaryotic DNA topoisomerase II, such as bisdioxopiperazines, or by trapping the covalent enzyme- DNA intermediate, such as epipodophyllotoxins. For mammalian DNA topoisomerase Iibeta and the newly discovered mammalian enzymes IIIalpha and IIIbeta, the physiological roles of which are yet to be defined, emphasis will be placed on the development of mouse models and cell lines. Deletion mutations have recently been introduced into mouse TOP2beta and TOP3alpha genes encoding DNA topoisomerases Iibeta and IIIalpha, and histopathological and biochemical studies of wild-type and heterozygous and homozygous mutant animals or cell lines will be carried out to deduce the physiological functions of these enzymes. Similar studies will be carried out with DNA topoisomerase IIIbeta. Cell lines derived from TOP2beta+/+ and top2Beta-/- embryos will also be used in comparative studies of anticancer drugs targeting the type II topoisomerases, so as to specifically assess the role of DNA topoisomerase IIbeta in cancer chemotherapy. The DNA topoisomerases are unique in that they are not only targets in the treatment of cancer, they may also play key roles in oncogenesis. The proposed studies may thus contribute to both cancer treatment and prevention.
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