A Unique Class of Topoisomerase Mutants That Are Hypersensitive to Multiple Antitumor Agents
Annual summary rept. 1 Mar 2000-28 Feb 2003
DUKE UNIV MEDICAL CENTER DURHAM NC
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The goal of this research is to understand the detailed mechanism of action of antitumor drugs that target type II topoisomerases. 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 G269V substitution suppresses a topoisomerase negative phenotype caused by the S79F substitution while the G269V substitution by itself was shown to confer hypersensitivity in vivo Cancer Research 58, 1260-1267. In order to understand these phenotypes on a biochemical level, I purified the S79F and G269V single mutant enzymes as well as the S79FG269V double mutant enzyme. I found the G269V enzyme to be hypersensitive to a number of cleavage-inducing antitumor agents and it 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. The S79F single and S79FG269V double mutant enzymes were resistant to the tested drugs. This resistance is likely due to an altered drug-binding pocket created by the S79F substitution. Unexpectedly, the S79F mutant enzyme has a defect in ATP dependence that is suppressed by the G269V substitution.
- Medicine and Medical Research