Antitumour drugs impede DNA uncoiling by topoisomerase I

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Abstract
Topoisomerases are enzymes that act to relax supercoiling, a form of built-up strain in DNA. Topoisomerase inhibitors are important antitumour drugs, thought to act by stabilizing a covalent complex between the topoisomerase and DNA, which then sets up a road-block to the DNA replication machinery. The origin of drug efficacy in targeting topoisomerases remains poorly understood, but now a singlemolecule study of the interaction of topotecan, a drug used mainly to treat ovarian cancer and small-cell lung cancer, and a topoisomerase IB–DNA complex, has revealed more details of the process. As illustrated on the cover, positive DNA supercoils accumulate due to the action of the drug (shown red) Such overwinding of the DNA hinders an advancing DNA polymerase and may play a role in the stalling or collapse of a replication fork, ultimately leading to cell death. A report on single topoisomerase molecules in action, showing that topoisomerase inhibitors don't just stabilize an intermediate but actually affect the ability of topoisomerase to relax DNA supercoiling, suggesting a new mechanism by which these drugs cause cytotoxicity. Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils1,2,3,4. Topotecan is thought to stabilize a covalent topoisomerase–DNA complex5,6,7, rendering it an obstacle to DNA replication forks2,3,8,9. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.

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