A Unique Class of Topoisomerase Mutants That Are Hypersensitive to Multiple Antitumor Agents

Abstract

Abstract : The goal of this research is to understand the detailed mechanism of action of antitumor drugs that target type II topoisomerases. Bacteriophage T4 provides a useful model system for the study of these antitumor agents. Previous analysis showed that a drug resistant bacteriophage T4 mutant harbored two amino acid substitutions (S79F, G269V) in topoisomerase subunit gp52. When both mutations are present, the O269V substitution suppresses a topoisomerase negative phenotype caused by the S79F substitution while the O269V substitution by itself was shown to confer hypersensitivity in vivo (Cancer Research 58, 1260-1267). 1 purified the G269V enzyme and found it to be hypersensitive to a number of cleavage-inducing antitumor agents. The G269V enzyme displayed an apparent 10-fold increase in drug-independent DNA cleavage, suggesting a novel mechanism of sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex. I have also purified the S79F mutant enzyme and am in the process of characterizing the defect caused by this substitution. The O269V mutant defines a new category of type II topoisomerase mutants, namely those that are hypersensitive to all inhibitors that stabilize the cleavage complex, and thereby offers new insights into the mechanism of drug action.