The bacteriophage T4 provides a powerful model system for analyzing the mechanism of action of antitumor agents that inhibit type II DNA topoisomerases. This relatively simple bacterial virus has been a favored organism for study since the early days of molecular biology, and is therefore very well characterized and easily manipulated. In spite of the prokaryotic nature of T4, the virus-encoded type II topoisomerase is inhibited by many of the same antitumor agents that inhibit the mammalian type II topoisomerase, including m-AMSA, mitoxantrone, ellipticines and epipodophyllotoxins. The first major goal of this proposal is to understand the nature of the inhibitor binding site in the topoisomerase- DNA complex. Several experiments are designed to test a working model in which the inhibitors intercalate precisely at the two DNA sites where the enzyme mediates phosphodiester bond cleavage. Regardless of whether the model is correct, these experiments should localize the site of inhibitor binding, clarify the rules of DNA sequence recognition by both enzyme and inhibitor, test the requirement for both enzyme and DNA in inhibitor binding, and analyze the binding of inhibitors to model substrates in the absence of enzyme. A second goal is to determine the mechanism of drug resistance conferred by mutational alterations in the topoisomerase. Two m-AMSA-resistant mutant enzymes will be analyzed to determine whether the drug-resistance mutation abolishes binding of the inhibitor. In addition, the amino acid substitutions in the two mutant enzymes will be deduced, and the altered rules of DNA sequence recognition by the mutant enzymes will be explored. The third major goal is to understand the recombinational repair of inhibitor-induced cleavage complexes. This repair pathway greatly reduces the drug sensitivity of wild type organisms, and is probably a key factor in the effectiveness of the antitumor agents under consideration. Using well-characterized T4 mutants, the roles of particular proteins in the repair pathway will be determined. New phage mutants with defects in recombinational repair will also be isolated, and genetic and physical tests will probe the products and intermediates in the recombinational repair pathway. Although these experiments will be conducted in a simple prokaryotic model system, the results are expected to contribute significantly to our understanding of antitumor drug action, and may suggest important new strategies for improving the therapeutic effectiveness of topoisomerase inhibitors.
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