Bacteriophage T4 provides a powerful model system for analyzing the mechanism of antitumor agents that inhibit type II DNA topoisomerases. T4 has been very well studied and is easy to manipulate with genetic, molecular and biochemical approaches. In spite of the prokaryotic nature of T4, the T4 encoded type II topoisomerase is sensitive to many of the antitumor agents that inhibit the mammalian enzyme, including m-AMSA, mitoxantrone, ellipticines and epipodophyllotoxins. Furthermore, the mechanism of inhibition of the T4 and mammalian enzymes appears to be identical. The proposed studies focus on the detailed mechanism of inhibition and the pathways of cytotoxicity and DNA repair that occur when cells are treated with the antitumor agents. We (and others) have extensively analyzed drug-resistant mutant topoisomerases. In the next grant period, we will analyze two different classes of mutants. We recently isolated a novel mutant that appears hypersensitive to all inhibitors that induce the cleavage complex, and we will conduct a systematic search for additional hypersensitive mutants. In addition, we will attempt to isolate mutants that behave as if the drug is always present. Biochemical analyses of these two classes should clarify both the mechanism of inhibition and the physiological consequences of topoisomerase inhibition. Our recent experiments have localized the drug at the DNA molecule in the active site of the enzyme. We propose to map the regions of protein that come into close contact with the drug-DNA complex. This analysis should be particularly informative, because crystal structures of two different conformations of type II topoisomerase have recently been published. Finally, we will also continue our studies of the mechanism of toxicity caused by topoisomerase inhibitors, and the pathway of recombinational repair that can mitigate this toxicity. Although these experiments are conducted in a prokaryotic model system, the results should advance our understanding of antitumor drug action and hopefully suggest new strategies for improving the therapeutic effectiveness of topoisomerase inhibitors.
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