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The type I DNA topoisomerases are targets for a variety of chemotherapeutic agents, Includin the antibacterial quinolones and several famile of antitumor drugs. These agents stabilize an epzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drugstabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons. The type II topoisomerases are a primary cellular target for many of the chemotherapeutic drugs described above. Studies on drug-resistant mammalian cell lines revealed alterations in topoisomerase levels or activity, suggesting that the enzyme is an important component of the cytotoxic response (6, 7). Genetic studies in Saccharomyces cerevisiae have demonstrated that a temperature-sensitive mutation in the type II topoisomerase gene greatly decreased drug sensitivity at the semipermissive temperature, presumably due to a reduction in topoisomerase activity (8). Perhaps the clearest evidence for a type II topoisomerase being the primary drug target comes from the T4 bacteriophage system. The properties of the T4 topoisomerase closely resemble those of the mammalian enzyme (1, 9). Most importantly, both are sensitive to the aminoacridine m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] (10, 11). A point mutation in one of the genes encoding the bacteriophage T4 topoisomerase allows drug-resistant phage growth, and topoisomerase purified from the mutant phage has drug-resistant topoisomerase activity (12,13).Topoisomerase inhibitors convert the enzymes that they target into poisons by inducing formation of the cleavage complex, a form of DNA damage. This view was first articulated when it was found that bacteriophage 17 12031The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The bacteriophage T4-encoded type II DNA topoisomerase is the major target for the antitumour agent m-AMSA (4'-(9-acridinylamino)methanesulphonm-ansidide) in phage-infected bacterial cells. Inhibition of the purified enzyme by m-AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double-strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase-deficient mutants were shown to be resistant to m-AMSA, indicating that m-AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m-AMSA, strongly suggesting that recombination participates in the repair of topoisomerase-mediated damage. Third, m-AMSA stimulated recombination in phage-infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m-AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage-infected cells.