Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

DNA cleavage assay for the identification of topoisomerase I inhibitors

Nature Protocols volume 3, pages 1736–1750 (2008)Cite this article

Abstract

The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach in the design of anticancer agents. However, despite the clinical successes of the camptothecin derivatives, a significant need for less toxic and more chemically stable Top1 inhibitors still persists. Here, we describe one of the most frequently used protocols to identify novel Top1 inhibitors. These methods use uniquely 3′-radiolabeled DNA substrates and denaturing polyacrylamide gel electrophoresis to provide evidence for the Top1-mediated DNA cleaving activity of potential Top1 inhibitors. These assays allow comparison of the effectiveness of different drugs in stabilizing the Top1-DNA intermediate or cleavage (cleavable) complex. A variation on these assays is also presented, which provides a suitable system for determining whether the inhibitor blocks the forward cleavage or religation reactions by measuring the reversibility of the drug-induced Top1–DNA cleavage complexes. This entire protocol can be completed in ∼2 d.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Structure and mechanism of the Top1 cleavage complex (Top1cc) trapping by a specific Top1 inhibitor (indenoisoquinoline derivative)4,5,55.
Figure 3: Structures of camptothecin derivatives.
Figure 4: Structures of the three chemical families of noncamptothecin Top1 inhibitors.
Figure 5: Cartoon illustrating the advantages of using a 3′-end-labeled DNA substrate.
Figure 6: Top1-mediated DNA cleavage assay.
Figure 7: Top1–DNA cleavage complex reversal assay.

Similar content being viewed by others

References

  1. Kohlhagen, G., Paull, K., Cushman, M., Nagafufuji, P. & Pommier, Y. Protein-linked DNA strand breaks induced by NSC 314622, a non-camptothecin topoisomerase I poison. Mol. Pharmacol. 54, 50–58 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Strumberg, D. et al. Synthesis of cytotoxic indenoisoquinoline topoisomerase I poisons. J. Med. Chem. 42, 446–457 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Antony, S. et al. Cellular Topoisomerase I Inhibition and antiproliferative activity by MJ-III-65 (NSC 706744), an indenoisoquinoline topoisomerase I poison. Mol. Pharmacol. 67, 523–530 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Ioanoviciu, A. et al. Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I poisons reveal an inhibitor with a flipped orientation in the ternary DNA-enzyme-inhibitor complex as determined by X-ray crystallographic analysis. J. Med. Chem. 48, 4803–4814 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Staker, B.L. et al. Structures of three classes of anticancer agents bound to the human topoisomerase I-DNA covalent complex. J. Med. Chem. 48, 2336–2345 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Antony, S. et al. Novel indenoisoquinolines NSC 725776 and NSC 724998 produce persistent topoisomerase I cleavage complexes and overcome multidrug resistance. Cancer Res. 67, 10397–10405 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Tanizawa, A., Kohn, K.W., Kohlhagen, G., Leteurtre, F. & Pommier, Y. Differential stabilization of eukaryotic DNA topoisomerase I cleavable complexes by camptothecin derivatives. Biochemistry 34, 7200–7206 (1995).

    Article  CAS  PubMed  Google Scholar 

  8. Pommier, Y. et al. Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites. Proc. Natl. Acad. Sci. USA 92, 8861–8865 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Urasaki, Y., Takebayashi, Y. & Pommier, Y. Activity of a novel camptothecin analogue, homocamptothecin, in camptothecin-resistant cell lines with topoisomerase I alterations. Cancer Res. 60, 6577–6580 (2000).

    CAS  PubMed  Google Scholar 

  10. Gamcsik, M.P. et al. Dual role of glutathione in modulating camptothecin activity: depletion potentiates activity, but conjugation enhances the stability of the topoisomerase I-DNA cleavage complex. Mol. Cancer Ther. 1, 11–20 (2001).

    CAS  PubMed  Google Scholar 

  11. Takagi, K. et al. Novel E-ring camptothecin keto analogues (S38809 and S39625) are stable, potent, and selective topoisomerase I inhibitors without being substrates of drug efflux transporters. Mol. Cancer Ther. 6, 3229–3238 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Morham, S., Kluckman, K.D., Voulomanos, N. & Smithies, O. Targeted disruption of the mouse topoisomerase I gene by camptothecin selection. Mol. Cell. Biol. 16, 6804–6809 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, C.X., Chen, A.D., Gettel, N.J. & Hsieh, T.S. Essential functions of DNA topoisomerase I in Drosophila melanogaster . Dev. Biol. 222, 27–40 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Christman, M.F., Dietrich, F.S. & Fink, G.R. Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerase I and II. Cell 55, 413–425 (1988).

    Article  CAS  PubMed  Google Scholar 

  15. Zhang, H. et al. Human mitochondrial topoisomerase I. Proc. Natl. Acad. Sci. USA 98, 10608–10613 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhang, H., Meng, L.H., Zimonjic, D.B., Popescu, N.C. & Pommier, Y. Thirteen-exon-motif signature for vertebrate nuclear and mitochondrial type IB topoisomerases. Nucleic Acids Res. 32, 2087–2092 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. Stewart, L., Redinbo, M.R., Qiu, X., Hol, W.G.J. & Champoux, J.J. A model for the mechanism of human topoisomerase I. Science 279, 1534–1541 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Lesher, D.T., Pommier, Y., Stewart, L. & Redinbo, M.R. 8-Oxoguanine rearranges the active site of human topoisomerase I. Proc. Natl. Acad. Sci. USA 99, 12102–12107 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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  CAS  PubMed  Google Scholar 

  24. Leteurtre, F. et al. Effects of DNA methylation on topoisomerase I and II cleavage activities. J. Biol. Chem. 269, 7893–7900 (1994).

    CAS  PubMed  Google Scholar 

  25. Henningfeld, K.A. & Hecht, S. A model for topoisomerase I-mediated insertions and deletions with duplex DNA substrates containing branches, nicks, and gaps. Biochemistry 34, 6120–6129 (1995).

    Article  CAS  PubMed  Google Scholar 

  26. Yeh, Y.-C., Liu, H.-F., Ellis, C.A. & Lu, A.-L. Mammalian topoisomerase I has a mismatch nicking activity. J. Biol. Chem. 269, 15498–15504 (1994).

    CAS  PubMed  Google Scholar 

  27. Pourquier, P. et al. Trapping of mammalian topoisomerase I and recombinations induced by damaged DNA containing nicks or gaps: importance of DNA end phosphorylation and camptothecin effects. J. Biol. Chem. 272, 26441–26447 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Pourquier, P. et al. Effects of uracil incorporation, DNA mismatches, and abasic sites on cleavage and religation activities of mammalian topoisomerase I. J. Biol. Chem. 272, 7792–7796 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Pourquier, P., Bjornsti, M.A. & Pommier, Y. Induction of topoisomerase I cleavage complexes by the vinyl chloride adduct 1,N6-ethenoadenine. J. Biol. Chem. 273, 27245–27249 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Takebayashi, Y., Pourquier, P., Yoshida, A., Kohlhagen, G. & Pommier, Y. Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove. Proc. Natl. Acad. Sci. USA 96, 7196–7201 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pourquier, P. et al. Gemcitabine (2′,2′-difluoro-2′-deoxycytidine), an antimetabolite that poisons topoisomerase I. Clin. Cancer Res. 8, 2499–2504 (2002).

    CAS  PubMed  Google Scholar 

  32. Pourquier, P. & Pommier, Y. Topoisomerase I-mediated DNA damage. Adv. Cancer Res. 80, 189–216 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Pourquier, P. et al. Topoisomerase I-mediated cytotoxicity of N-methyl-N′-nitro-N-nitrosoguanidine: trapping of topoisomerase I by the O6-methylguanine. Cancer Res. 61, 53–58 (2001).

    CAS  PubMed  Google Scholar 

  34. Dexheimer, T.S., Kozekova, A., Rizzo, C.J., Stone, M.P. & Pommier, Y. The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts. Nucleic Acids Res. 36, 4128–4136 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lanza, A., Tornatelli, S., Rodolfo, C., Scanavini, M.C. & Pedrini, A.M. Human DNA topoisomerase I-mediated cleavages stimulated by ultraviolet light-induced DNA damage. J. Biol. Chem. 271, 6978–6986 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Subramanian, D., Rosenstein, B.S. & Muller, M.T. Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair. Cancer Res. 58, 976–984 (1998).

    CAS  PubMed  Google Scholar 

  37. Chrencik, J.E., Burgin, A.B., Pommier, Y., Stewart, L. & Redinbo, M.R. Structural impact of the leukemia drug Ara-C on the covalent human topoisomerase I DNA complex. J. Biol. Chem. 278, 12461–12466 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Antony, S., Arimondo, P.B., Sun, J.S. & Pommier, Y. Position- and orientation-specific enhancement of topoisomerase I cleavage complexes by triplex DNA structures. Nucleic Acids Res. 32, 5163–5173 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Antony, S. et al. Enhancement of camptothecin-induced topoisomerase I cleavage complexes by the acetaldehyde adduct N2-ethyl-2′-deoxyguanosine. Nucleic Acids Res. 32, 5685–5692 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Pommier, Y. et al. Different effects on human topoisomerase I by minor groove and intercalated deoxyguanosine adducts derived from two polycyclic aromatic hydrocarbon diol epoxides at or near a normal cleavage site. J. Biol. Chem. 277, 13666–13672 (2002).

    Article  CAS  PubMed  Google Scholar 

  41. Pommier, Y. et al. Benzo[a]pyrene epoxide adducts in DNA are potent inhibitors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages. Proc. Natl. Acad. Sci. USA 97, 2040–2045 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pommier, Y. et al. Position-specific trapping of topoisomerase I-DNA cleavage complexes by intercalated benzo[a]- pyrene diol epoxide adducts at the 6-amino group of adenine. Proc. Natl. Acad. Sci. USA 97, 10739–10744 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sordet, O. et al. Apoptotic topoisomerase I-DNA complexes induced by staurosporine-mediated oxygen radicals. J. Biol. Chem. 279, 50499–50504 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Sordet, O., Khan, Q.A. & Pommier, Y. Apoptotic topoisomerase I-DNA complexes induced by oxygen radicals and mitochondrial dysfunction. Cell Cycle 3, 1095–1097 (2004).

    Article  CAS  PubMed  Google Scholar 

  45. Sordet, O. et al. Topoisomerase I-DNA complexes contribute to arsenic trioxide-induced apoptosis. J. Biol. Chem. 279, 33968–33975 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Soe, K., Rockstroh, A., Schache, P. & Grosse, F. The human topoisomerase I damage response plays a role in apoptosis. DNA Repair (Amst.) 3, 387–393 (2004).

    Article  CAS  Google Scholar 

  47. Rockstroh, A., Kleinert, A., Kramer, M., Grosse, F. & Soe, K. Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner. Oncogene 26, 123–131 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Sordet, O., Goldman, A. & Pommier, Y. Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes. Mol. Cancer Ther. 5, 3139–3144 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Ganguly, A. et al. Betulinic acid, a catalytic inhibitor of topoisomerase I, inhibits reactive oxygen species-mediated apoptotic topoisomerase I-DNA cleavable complex formation in prostate cancer cells but does not affect the process of cell death. Cancer Res. 67, 11848–11858 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Sen, N. et al. Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex. Cell Death Differ. 14, 358–367 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Sordet, O. et al. Topoisomerase I requirement for death receptor-induced apoptotic nuclear fission. J. Biol. Chem. 283, 23200–23208 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Pommier, Y. et al. Repair of topoisomerase I-mediated DNA damage. Prog. Nucleic Acid Res. Mol. Biol. 81, 179–229 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Bertrand, R., Solary, E., Kohn, K.W., O'Connor, P. & Pommier, Y. Induction of a common pathway to apoptosis by staurosporine. Exp. Cell Res. 211, 314–321 (1994).

    Article  CAS  PubMed  Google Scholar 

  54. Pommier, Y. & Cherfils, J. Interfacial protein inhibition: a nature's paradigm for drug discovery. Trends Pharmacol. Sci. 28, 136–145 (2005).

    Google Scholar 

  55. Marchand, C. et al. A novel norindenoisoquinoline structure reveals a common interfacial inhibitor paradigm for ternary trapping of topoisomerase I-DNA covalent complexes. Mol. Cancer Ther. 5, 287–295 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Nitiss, J. & Wang, J.C. DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc. Natl. Acad. Sci. USA 85, 7501–7505 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Bjornsti, M.-A., Benedetti, P., Viglianti, G.A. & Wang, J.C. Expression of human DNA topoisomerase I in Yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the antitumor drug camptothecin. Cancer Res. 49, 6318–6323 (1989).

    CAS  PubMed  Google Scholar 

  58. Wall, M.E. & Wani, M.C. Camptothecin and taxol: discovery to clinic—thirteenth Bruce F. Cain Memorial Award lecture. Cancer Res. 55, 753–760 (1995).

    CAS  PubMed  Google Scholar 

  59. Sirikantaramas, S., Yamazaki, M. & Saito, K. Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants. Proc. Natl. Acad. Sci. USA 105, 6782–6786 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Fujimori, A., Harker, W.G., Kohlhagen, G., Hoki, Y. & Pommier, Y. Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell resistant to camptothecin. Cancer Res. 55, 1339–1346 (1995).

    CAS  PubMed  Google Scholar 

  61. 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 

  62. Teicher, B.A. Next generation topoisomerase I inhibitors: rationale and biomarker strategies. Biochem. Pharmacol. 75, 1262–1271 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. Pratesi, G., Beretta, G.L. & Zunino, F. Gimatecan, a novel camptothecin with a promising preclinical profile. Anticancer Drugs 15, 545–552 (2004).

    Article  CAS  PubMed  Google Scholar 

  64. Jaxel, C., Kohn, K.W., Wani, M.C., Wall, M.E. & Pommier, Y. Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: evidence for a specific receptor site and a relation to antitumor activity. Cancer Res. 49, 1465–1469 (1989).

    CAS  PubMed  Google Scholar 

  65. Hsiang, Y.H. et al. DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res. 49, 4385–4389 (1989).

    CAS  PubMed  Google Scholar 

  66. Mi, Z. & Burke, T.G. Differential interactions of camptothecin lactone and carboxylate forms with human blood components. Biochemistry 33, 10325–10336 (1994).

    Article  CAS  PubMed  Google Scholar 

  67. Strumberg, D. et al. Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5′-phosphorylated DNA double-strand breaks by replication runoff. Mol. Cell Biol. 20, 3977–3987 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Lavergne, O. et al. Homocamptothecins: synthesis and antitumor activity of novel E-ring-modified camptothecin analogues. J. Med. Chem. 41, 5410–5419 (1998).

    Article  CAS  PubMed  Google Scholar 

  69. Bailly, C. Homocamptothecins: potent topoisomerase I inhibitors and promising anticancer drugs. Crit. Rev. Oncol. Hematol. 45, 91–108 (2003).

    Article  PubMed  Google Scholar 

  70. Hautefaye, P. et al. Synthesis and pharmacological evaluation of novel non-lactone analogues of camptothecin. Bioorg Med. Chem. Lett. 13, 2731–2735 (2003).

    Article  CAS  PubMed  Google Scholar 

  71. Meng, L.-H., Liao, Z.-Y. & Pommier, Y. Non-camptothecin DNA topoisomerase I inhibitors in cancer chemotherapy. Curr. Topics Med. Chem. 3, 305–320 (2003).

    Article  CAS  Google Scholar 

  72. Bailly, C. Topoisomerase I poisons and suppressors as anticancer drugs. Curr. Med. Chem. 7, 39–58 (2000).

    Article  CAS  PubMed  Google Scholar 

  73. Paull, K.D. et al. Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of a mean graph and COMPARE algorithm. J. Natl. Cancer Inst. 81, 1088–1092 (1989).

    Article  CAS  PubMed  Google Scholar 

  74. Yamashita, Y. et al. Induction of mammalian DNA topoisomerase I-mediated DNA cleavage by antitumor indolocarbazole derivatives. Biochemistry 31, 12069–12075 (1992).

    Article  CAS  PubMed  Google Scholar 

  75. Long, B.H., Rose, W.C., Vyas, D.M., Matson, J.A. & Forenza, S. Discovery of antitumor indolocarbazoles: rebeccamycin, NSC 655649, and fluoroindolocarbazoles. Curr. Med. Chem. Anticancer Agents 2, 255–266 (2002).

    Article  CAS  PubMed  Google Scholar 

  76. Li, T.K. et al. Characterization of ARC-111 as a novel topoisomerase I-targeting anticancer drug. Cancer Res. 63, 8400–8407 (2003).

    CAS  PubMed  Google Scholar 

  77. Morrell, A., Antony, S., Kohlhagen, G., Pommier, Y. & Cushman, M. A systematic study of nitrated indenoisoquinolines reveals a potent topoisomerase I inhibitor. J. Med. Chem. 49, 7740–7753 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Antony, S. et al. Differential induction of topoisomerase I-DNA cleavage complexes by the indenoisoquinoline MJ-III-65 (NSC 706744) and camptothecin: base sequence analysis and activity against camptothecin-resistant topoisomerases I. Cancer Res. 63, 7428–7435 (2003).

    CAS  PubMed  Google Scholar 

  79. Morrell, A. et al. Evaluation of indenoisoquinoline topoisomerase I inhibitors using a hollow fiber assay. Bioorg. Med. Chem. 16, 4395–4399 (2006).

    Article  CAS  Google Scholar 

  80. Morrell, A. et al. Nitrated indenoisoquinolines as topoisomerase I inhibitors: a systematic study and optimization. J. Med. Chem. 50, 4419–4430 (2007).

    Article  CAS  PubMed  Google Scholar 

  81. Morrell, A. et al. Investigation of the lactam side chain length necessary for optimal indenoisoquinoline topoisomerase I inhibition and cytotoxicity in human cancer cell cultures. J. Med. Chem. 50, 2040–2048 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Svejstrup, J.Q., Christiansen, K., Andersen, A.H., Lund, K. & Westergaard, O. Minimal DNA duplex requirements for topoisomerase I-mediated cleavage in vitro . J. Biol. Chem. 265, 12529–12535 (1990).

    CAS  PubMed  Google Scholar 

  83. Bonven, B.J., Gocke, E. & Westergaard, O. A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin. Cell 41, 541–551 (1985).

    Article  CAS  PubMed  Google Scholar 

  84. Christiansen, K., Svejstrup, A.B., Andersen, A.H. & Westergaard, O. Eukaryotic topoisomerase I-mediated cleavage requires bipartite DNA interaction. Cleavage of DNA substrates containing strand interruptions implicates a role for topoisomerase I in illegitimate recombination. J. Biol. Chem. 268, 9690–9701 (1993).

    CAS  PubMed  Google Scholar 

  85. Jaxel, C., Capranico, G., Kerrigan, D., Kohn, K.W. & Pommier, Y. Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin. J. Biol. Chem. 266, 20418–20423 (1991).

    CAS  PubMed  Google Scholar 

  86. Antony, S. et al. Bisindenoisoquinoline bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propyla mino}propane bis(trifluoroacetate) (NSC 727357), a DNA intercalator and topoisomerase inhibitor with antitumor activity. Mol. Pharmacol. 70, 1109–1120 (2006).

    Article  CAS  PubMed  Google Scholar 

  87. Bjornsti, M.-A. & Osheroff, N. (eds.) DNA topoisomerase protocols. DNA topology and enzymes Vol. 94 (Humana Press, Totowa, NJ, 1999).

  88. Pommier, Y., Covey, J.M., Kerrigan, D., Markovits, J. & Pham, R. DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators. Nucleic Acids Res. 15, 6713–6731 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Jaxel, C., Kohn, K.W. & Pommier, Y. Topoisomerase I interaction with SV40 DNA in the presence and absence of camptothecin. Nucleic Acids Res. 16, 11157–11170 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. McClendon, A.K. & Osheroff, N. The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs. Biochemistry 45, 3040–3050 (2006).

    Article  CAS  PubMed  Google Scholar 

  91. Kohn, K.W. DNA filter elution: a window on DNA damage in mammalian cells. Bioessays 18, 505–513 (1996).

    Article  CAS  PubMed  Google Scholar 

  92. Trask, D.K., DiDonato, J.A. & Muller, M.T. Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II. Embo. J. 3, 671–676 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. El-Khamisy, S.F. et al. Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1. Nature 434, 108–113 (2005).

    Article  CAS  PubMed  Google Scholar 

  94. Laco, G.S. et al. Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies. Bioorg. Med. Chem. 12, 5225–5235 (2004).

    Article  CAS  PubMed  Google Scholar 

  95. Maxam, A. & Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Meth. Enzymol. 65, 499–560 (1980).

    Article  CAS  Google Scholar 

  96. Pommier, Y., Pourquier, P., Urasaki, Y., Wu, J. & Laco, G.S. Topoisomerase I inhibitors: selectivity and cellular resistance. Drug Resist. Updat. 2, 307–318 (1999).

    Article  CAS  PubMed  Google Scholar 

  97. Yoshinari, T. et al. Mode of action of a new indolocarbazole anticancer agent, J-107088, targeting topoisomerase I. Cancer Res. 59, 4271–4275 (1999).

    CAS  PubMed  Google Scholar 

  98. Zhu, S. et al. Esters and amides of 2,3-dimethoxy-8,9-methylenedioxy-benzo[i]phenanthridine-12-carboxylic acid: potent cytotoxic and topoisomerase I-targeting agents. Bioorg. Med. Chem. 13, 6782–6794 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Our studies are supported by the Intramural Program of the Center for Cancer Research, NCI, NIH. We thank all members of the Laboratory of Molecular Pharmacology, past and present, for their contributions. Special thanks to Dr Kurt W. Kohn who pioneered studies on DNA topoisomerases and first proposed the trapping of topoisomerases by anticancer drugs (Ross, W.E., Glaubiger, D. & Kohn, K.W. Qualitative and quantitative aspects of intercalator-induced DNA strand breaks. Biochim. Biophys. Acta 562, 41–50 (1979)).

Author information

Authors and Affiliations

  1. Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Thomas S Dexheimer & Yves Pommier

Authors
  1. Thomas S Dexheimer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yves Pommier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yves Pommier.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dexheimer, T., Pommier, Y. DNA cleavage assay for the identification of topoisomerase I inhibitors. Nat Protoc 3, 1736–1750 (2008). https://doi.org/10.1038/nprot.2008.174

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.174

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing