DNA Topoisomerases pp 119-130

Part of the Methods in Molecular Biology™ book series (MIMB, volume 582)

Analyzing Top2 Distribution on Yeast Chromosomes by Chromatin Immunoprecipitation

  • Melissa Baldwin
  • Tariq Warsi
  • Jeff Bachant
Protocol

Abstract

In vertebrate cells, DNA topoisomerase II (Topo II), named Top2 in yeast, localizes along chromosome axes early in mitosis and concentrates within centromeric chromatin during metaphase. The factors controlling these changes in enzyme distribution are largely unknown. Insight into Topo II dynamics could potentially be derived through genetic approaches in yeast. In practice, however, the small size and limited compaction of yeast chromosomes has precluded a detailed analysis of Top2 localization along mitotic chromosomes. As an alternative approach, we describe a method for examining Top2 distribution using chromatin immunoprecipitation (ChIP). By adding a detergent solubilization step, this method allows efficient recovery of DNA sequences associated with Top2 in the insoluble chromosome scaffold fraction.

Key words

DNA topoisomerase II (Top2) chromatin immunoprecipitation (ChIP) 

References

  1. 1.
    Earnshaw, W. C., Halligan, B., Cooke, C. A., Heck, M. M. and Liu, L. F. (1985) Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol 100, 1706–15.PubMedCrossRefGoogle Scholar
  2. 2.
    Gasser, S. M., Laroche, T., Falquet, J., Boy de la Tour, E. and Laemmli, U. K. (1986) Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol 188, 613–29.PubMedCrossRefGoogle Scholar
  3. 3.
    Belmont, A. S. (2002) Mitotic chromosome scaffold structure: new approaches to an old controversy. Proc Natl Acad Sci USA 99, 15855–7.PubMedCrossRefGoogle Scholar
  4. 4.
    Christensen, M. O., Larsen, M. K., Barthelmes, H. U., Hock, R., Andersen, C. L., Kjeldsen, E., Knudsen, B. R., Westergaard, O., Boege, F. and Mielke, C. (2002) Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells. J Cell Biol 157, 31–44.PubMedCrossRefGoogle Scholar
  5. 5.
    Tavormina, P. A., Come, M. G., Hudson, J. R., Mo, Y. Y., Beck, W. T. and Gorbsky, G. J. (2002) Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis. J Cell Biol 158, 23–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Porter, A. C. and Farr, C. J. (2004) Topoisomerase II: untangling its contribution at the centromere. Chromosome Res 12, 569–83.PubMedCrossRefGoogle Scholar
  7. 7.
    Bermejo, R., Doksani, Y., Capra, T., Katou, Y. M., Tanaka, H., Shirahige, K. and Foiani, M. (2007) Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21, 1921–36.PubMedCrossRefGoogle Scholar
  8. 8.
    Takahashi, Y., Yong-Gonzalez, V., Kikuchi, Y. and Strunnikov, A. (2006) SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II. Genetics 172, 783–94.PubMedCrossRefGoogle Scholar
  9. 9.
    Sikorski, R. S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.PubMedGoogle Scholar
  10. 10.
    Strahl-Bolsinger, S., Hecht, A., Luo, K. and Grunstein, M. (1997) SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 11, 83–93.PubMedCrossRefGoogle Scholar
  11. 11.
    Meluh, P. B. and Koshland, D. (1997) Budding yeast centromere composition and assembly as revealed by in vivo cross-linking. Genes Dev 11, 3401–12.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Melissa Baldwin
    • 1
  • Tariq Warsi
    • 1
  • Jeff Bachant
    • 1
  1. 1.Department of Cell Biology and NeuroscienceUniversity of California at RiversideRiversideUSA

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