A unique type II topoisomerase mutant that is hypersensitive to a broad range of cleavage-inducing antitumor agents.

Abstract

Bacteriophage T4 provides a useful model system for dissecting the mechanism of action of antitumor agents that target type II DNA topoisomerases. Many of these inhibitors act by trapping the cleavage complex, a covalent complex of enzyme and broken DNA. Previous analysis showed that a drug-resistant T4 mutant harbored two amino acid substitutions (S79F, G269V) in topoisomerase subunit gp52. Surprisingly, the single amino acid substitution, G269V, was shown to confer hypersensitivity in vivo to m-AMSA and oxolinic acid [Freudenreich, C. H., et al. (1998) Cancer Res. 58, 1260-1267]. We purified this G269V mutant enzyme and found it to be hypersensitive to a number of cleavage-inducing inhibitors including m-AMSA, VP-16, mitoxantrone, ellipticine, and oxolinic acid. While the mutant enzyme did not exhibit altered DNA cleavage site specificity compared to the wild-type enzyme, it did display an apparent 10-fold increase in drug-independent DNA cleavage. This suggests a novel mechanism of altered drug sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex, resulting in an increase in the concentration of cleavage intermediates available to inhibitors. Mutations that alter drug sensitivities tend to cluster within two specific regions of all type II topoisomerases. Residue G269 of gp52 lies outside of these regions, and it is therefore not surprising that G269V leads to a unique mechanism of drug hypersensitivity. We believe that this mutant defines a new category of type II topoisomerase mutants, namely, those that are hypersensitive to all inhibitors that stabilize the cleavage complex.