The purpose of this proposed research is to study the molecular mechanism of cell killing by anti-cancer drugs which are known to produce protein-linked DNA breaks in vivo. Our long term goal is to understand the regulatory mechanism of cell proliferation and cell death. The proposed research focuses on the phenomenon of protein-linked DNA breaks which represents a novel type of DNA damage produced by many potent antitumor agents. Relatively little is known about the biological consequences of this particular DNA damage. Recently, there is increasing evidence that mammalian DNA topoisomerase II may be the intracellular target of these antitumor drugs and the formation of protein-linked DNA breaks may be the direct result of drug-topoisomerase or drug-topoisomerase-DNA interaction. In vitro studies have suggested that many antitumor drugs may interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by blocking the reunion of transiently broken DNA. Whether the observed in vitro reaction reflects drug action in vivo has not been fully established. Furthermore, the molecular mechanism of rapid cell killing by these antitumor drugs is still not known. In order to answer these questions, both genetic and biochemical experiments will be performed to identify mammalian DNA topoisomerase II as the cytotoxic target. Genetic analyses of drug resistant mutants will be used to identify mammalian topoisomerase II as the primary cytotoxic target of these antitumor drugs. Localization of the protein-linked DNA breaks on human chromosomes will be carried out by an indirect end labeling mapping procedure. The nucleotide sequences around the breakage sites will be determined by cloning and sequencing. DNA clones containing the strong breakage sites will be used for in vitro studies using purified HeLa topoisomerase II. Human cells treated with epipodophyllotoxin, VP-16, will be used as a model system for the analyses of the biological consequences leading to cell death.
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