Advertisement

Detection of Covalent DNA-Bound Spo11 and Topoisomerase Complexes

  • Edgar HartsuikerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

Abstract

Topoisomerases can release topological stress and resolve DNA catenanes by a DNA strand breakage and re-ligation mechanism. During the lifetime of the DNA break, the topoisomerase remains covalently linked to the DNA and removes itself when the break is re-ligated. While the lifetime of a covalent topoisomerase–DNA complex is usually short, several clinically important cancer drugs kill cancer cells by inhibiting the removal of covalently linked topoisomerases. The topoisomerase-like protein Spo11 is responsible for meiotic double strand break formation. Spo11 is not able to remove itself and is removed by nucleolytic cleavage. This chapter describes a method which allows the reproducible and quantitative detection of proteins covalently bound to the DNA.

Key words

Topoisomerase I topoisomerase II Spo11 Schizosaccharomyces pombe MRN complex Tdp1 

References

  1. 1.
    Champoux, J.J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70, 369–413.PubMedCrossRefGoogle Scholar
  2. 2.
    Pommier, Y. (2004) Camptothecins and topoisomerase I: a foot in the door. Targeting the genome beyond topoisomerase I with camptothecins and novel anticancer drugs: importance of DNA replication, repair and cell cycle checkpoints. Curr Med Chem Anticancer Agents 4, 429–434.PubMedCrossRefGoogle Scholar
  3. 3.
    Baldwin, E.L., and Osheroff, N. (2005) Etoposide, topoisomerase II and cancer. Curr Med Chem Anticancer Agents 5, 363–372.PubMedCrossRefGoogle Scholar
  4. 4.
    Pouliot, J.J., Yao, K.C., Robertson, C.A., and Nash, H.A. (1999) Yeast gene for a Tyr-DNA phosphodiesterase that repairs topoisomerase I complexes. Science 286, 552–555.PubMedCrossRefGoogle Scholar
  5. 5.
    Cortes Ledesma, F., El Khamisy, S.F., Zuma, M.C., Osborn, K., and Caldecott, K.W. (2009) A human 5-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature 461, 674–678.PubMedCrossRefGoogle Scholar
  6. 6.
    Connelly, J.C., and Leach, D.R.F. (2004) Repair of DNA covalently linked to protein. Mol Cell 13, 307–316.PubMedCrossRefGoogle Scholar
  7. 7.
    Neale, M.J., Pan, J., and Keeney, S. (2005) Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053–1057.PubMedCrossRefGoogle Scholar
  8. 8.
    Neale, M.J., and Keeney, S. (2009) End-labeling and analysis of Spo11-oligonucleotide complexes in Saccharomyces cerevisiae. Methods Mol Biol 557, 183–195.PubMedCrossRefGoogle Scholar
  9. 9.
    Hartsuiker, E., Mizuno, K., Molnar, M., Kohli, J., Ohta, K., and Carr, A.M. (2009) Ctp1CtIP and Rad32Mre11 nuclease activity are required for Rec12Spo11 removal, but Rec12Spo11 removal is dispensable for other MRN-dependent meiotic functions. Mol Cell Biol 29, 1671–1681.PubMedCrossRefGoogle Scholar
  10. 10.
    Hartsuiker, E., Neale, M.J., and Carr, A.M. (2009) Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA. Mol Cell 33, 117–123.PubMedCrossRefGoogle Scholar
  11. 11.
    Keeney, S., Giroux, C.N., and Kleckner, N. (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375–384.PubMedCrossRefGoogle Scholar
  12. 12.
    Shaw, J.L., Blanco, J., and Mueller, G.C. (1975) Simple procedure for isolation of DNA, RNA and protein fractions from cultured animal cells. Anal Biochem 65, 125–131.PubMedCrossRefGoogle Scholar
  13. 13.
    El-Khamisy, S.F., Hartsuiker, E., and Caldecott, K.W. (2007) TDP1 facilitates repair of ionizing radiation-induced DNA single-strand breaks. DNA Repair (Amst) 6, 1485–1495.CrossRefGoogle Scholar
  14. 14.
    Bähler, J., Schuchert, P., Grimm, C., and Kohli, J. (1991) Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells. Curr Genet 19, 445–451.PubMedCrossRefGoogle Scholar
  15. 15.
    Forsburg, S.L., and Rhind, N. (2006) Basic methods for fission yeast. Yeast 23, 173–183.PubMedCrossRefGoogle Scholar
  16. 16.
    Utsugi, T., Shibata, J., Sugimoto, Y., Aoyagi, K., Wierzba, K., Kobunai, T., Terada, T., Oh-hara, T., Tsuruo, T., and Yamada, Y. (1996) Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and lung metastatic cancer. Cancer Res 56, 2809–2814.PubMedGoogle Scholar
  17. 17.
    Cervantes, M.D., Farah, J.A., and Smith, G.R. (2000) Meiotic DNA breaks associated with recombination in S. pombe. Mol Cell 5, 883–888.PubMedCrossRefGoogle Scholar
  18. 18.
    Subramanian, D., Furbee, C.S., and Muller, M.T. (2001) ICE bioassay. Isolating in vivo complexes of enzyme to DNA. Methods Mol Biol 95, 137–147.PubMedGoogle Scholar
  19. 19.
    Chikashige, Y., Kurokawa, R., Haraguchi, T., and Hiraoka, Y. (2004) Meiosis induced by inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of centromeres in the fission yeast Schizosaccharomyces pombe. Genes Cells 9, 671–684.PubMedCrossRefGoogle Scholar
  20. 20.
    Bähler, J., Wyler, T., Loidl, J., and Kohli, J. (1993) Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis, J. Cell Biol 121, 241–256.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.North West Cancer Research Fund Institute, Bangor UniversityBangorUK

Personalised recommendations