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Antitumour drugs impede DNA uncoiling by topoisomerase I

Abstract

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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Figure 1: Drug-dependent slow DNA uncoiling by human TopIB.
Figure 2: The duration of slow removal and its uniqueness to the removal of positive supercoils.
Figure 3: Camptothecin poisoning of TopIB induces accumulation of positively supercoiled DNA in G1- and S-phase yeast cells.
Figure 4: Mechanism of camptothecin-induced toxicity derives from TopIB-dependent accumulation of positive supercoils.

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Acknowledgements

We thank K. Besteman and U. Keyser for discussions, S. Hage and Y.-H. Chien for DNA preparation, R. van Waardenburg for help with PyMol, and C. Dekker for critical reading and general support. We thank FOM, NWO, NIH and ALSAC for financial support.

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Correspondence to Nynke H. Dekker.

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Koster, D., Palle, K., Bot, E. et al. Antitumour drugs impede DNA uncoiling by topoisomerase I. Nature 448, 213–217 (2007). https://doi.org/10.1038/nature05938

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