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Antitumour drugs impede DNA uncoiling by topoisomerase I

Nature volume 448, pages 213–217 (2007)Cite this article

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.

References

  1. Champoux, J. J. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413 (2001)

    Article  CAS  Google Scholar 

  2. Li, T. K. & Liu, L. F. Tumor cell death induced by topoisomerase-targeting drugs. Annu. Rev. Pharmacol. Toxicol. 41, 53–77 (2001)

    Article  Google Scholar 

  3. Pommier, Y. Topoisomerase I inhibitors: camptothecins and beyond. Nature Rev. Cancer 6, 789–802 (2006)

    Article  CAS  Google Scholar 

  4. Wang, J. C. Cellular roles of DNA topoisomerases: a molecular perspective. Nature Rev. Mol. Cell Biol. 3, 430–440 (2002)

    Article  CAS  Google Scholar 

  5. Hsiang, Y. H., Hertzberg, R., Hecht, S. & Liu, L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem. 260, 14873–14878 (1985)

    CAS  PubMed  Google Scholar 

  6. Porter, S. E. & Champoux, J. J. The basis for camptothecin enhancement of DNA breakage by eukaryotic topoisomerase I. Nucleic Acids Res. 17, 8521–8532 (1989)

    Article  CAS  Google Scholar 

  7. Staker, B. L. et al. The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc. Natl Acad. Sci. USA 99, 15387–15392 (2002)

    Article  ADS  CAS  Google Scholar 

  8. Holm, C., Covey, J. M., Kerrigan, D. & Pommier, Y. Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells. Cancer Res. 49, 6365–6368 (1989)

    CAS  PubMed  Google Scholar 

  9. Hsiang, Y. H., Lihou, M. G. & Liu, L. F. Arrest of replication forks by drug-stabilized topoisomerase I–DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 49, 5077–5082 (1989)

    CAS  PubMed  Google Scholar 

  10. Corbett, K. D. & Berger, J. M. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 33, 95–118 (2004)

    Article  CAS  Google Scholar 

  11. Bjornsti, M. A. Cancer therapeutics in yeast. Cancer Cell 2, 267–273 (2002)

    Article  CAS  Google Scholar 

  12. Minsky, B. D. Combined-modality therapy of rectal cancer with irinotecan-based regimens. Oncology (Huntingt.) 18, 49–55 (2004)

    Google Scholar 

  13. Rodriguez-Galindo, C. et al. Clinical use of topoisomerase I inhibitors in anticancer treatment. Med. Pediatr. Oncol. 35, 385–402 (2000)

    Article  CAS  Google Scholar 

  14. Stewart, D. J. Topotecan in the first-line treatment of small cell lung cancer. Oncologist 9 (Suppl 6). 33–42 (2004)

    Article  CAS  Google Scholar 

  15. Sekiguchi, J. & Shuman, S. Vaccinia topoisomerase binds circumferentially to DNA. J. Biol. Chem. 269, 31731–31734 (1994)

    CAS  PubMed  Google Scholar 

  16. Redinbo, M. R., Stewart, L., Kuhn, P., Champoux, J. J. & Hol, W. G. Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279, 1504–1513 (1998)

    Article  ADS  CAS  Google Scholar 

  17. Koster, D. A., Croquette, V., Dekker, C., Shuman, S. & Dekker, N. H. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature 434, 671–674 (2005)

    Article  ADS  CAS  Google Scholar 

  18. Stivers, J. T., Harris, T. K. & Mildvan, A. S. Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation. Biochemistry 36, 5212–5222 (1997)

    Article  CAS  Google Scholar 

  19. Chrencik, J. E. et al. Mechanisms of camptothecin resistance by human topoisomerase I mutations. J. Mol. Biol. 339, 773–784 (2004)

    Article  CAS  Google Scholar 

  20. Strick, T., Allemand, J., Croquette, V. & Bensimon, D. Twisting and stretching single DNA molecules. Prog. Biophys. Mol. Biol. 74, 115–140 (2000)

    Article  CAS  Google Scholar 

  21. Champoux, J. J. & Aronoff, R. The effects of camptothecin on the reaction and the specificity of the wheat germ type I topoisomerase. J. Biol. Chem. 264, 1010–1015 (1989)

    CAS  PubMed  Google Scholar 

  22. Stewart, L., Ireton, G. C. & Champoux, J. J. A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay. J. Biol. Chem. 274, 32950–32960 (1999)

    Article  CAS  Google Scholar 

  23. Woo, M. H., Vance, J. R., Marcos, A. R., Bailly, C. & Bjornsti, M. A. Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin. J. Biol. Chem. 277, 3813–3822 (2002)

    Article  CAS  Google Scholar 

  24. Giaever, G. N. & Wang, J. C. Supercoiling of intracellular DNA can occur in eukaryotic cells. Cell 55, 849–856 (1988)

    Article  CAS  Google Scholar 

  25. Liu, L. F. & Wang, J. C. Supercoiling of the DNA template during transcription. Proc. Natl Acad. Sci. USA 84, 7024–7027 (1987)

    Article  ADS  CAS  Google Scholar 

  26. Goto, T. & Wang, J. C. Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell 36, 1073–1080 (1984)

    Article  CAS  Google Scholar 

  27. Woo, M. H. et al. Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality. Proc. Natl Acad. Sci. USA 100, 13767–13772 (2003)

    Article  ADS  CAS  Google Scholar 

  28. Strick, T. R., Allemand, J. F., Bensimon, D., Bensimon, A. & Croquette, V. The elasticity of a single supercoiled DNA molecule. Science 271, 1835–1837 (1996).

    Article  ADS  CAS  Google Scholar 

  29. Hann, C. et al. Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I. J. Biol. Chem. 273, 8425–8433 (1998)

    Article  CAS  Google Scholar 

  30. Megonigal, M. D., Fertala, J. & Bjornsti, M. A. Alterations in the catalytic activity of yeast DNA topoisomerase I result in cell cycle arrest and cell death. J. Biol. Chem. 272, 12801–12808 (1997)

    Article  CAS  Google Scholar 

Download references

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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Authors and Affiliations

  1. Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands,

    Daniel A. Koster, Elisa S. M. Bot & Nynke H. Dekker

  2. Department of Molecular Pharmacology, St Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105, USA,

    Komaraiah Palle & Mary-Ann Bjornsti

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  1. Daniel A. Koster
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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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