Skip to main content
SpringerLink
Log in

Overexpression and Purification of DNA Topoisomerase I from Yeast

  • Protocol
  • First Online:
DNA Topoisomerase Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 94))

  • 1038 Accesses

Abstract

The mechanism of action of DNA topoisomerase I in catalyzing the relaxation of supercoiled DNA and how this reaction is perturbed by the antitumor drug camptothecin have been the subject of intense investigation (reviewedin (1-3). Much of this effort has focused on structure/function studies of wild-type and mutant forms of the enzyme, derived from a variety of sources (1,4-20). The budding yeast Saccharomyces cerevisiae has proven particularly amenable to the genetic manipulations required to overexpress and purify wild-type and mutant forms of eukaryotic DNA topoisomerase I (3,4,12,13).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wang, J. C. (1996) DNA topoisomerases. Ann. Rev. Biochem. 65, 635–692.

    Article  CAS  Google Scholar 

  2. Chen, A. and Liu, L. F. (1994) DNA topoisomerases: essential enzymes and lethal targets. Ann. Rev. Pharmacol. Toxicol. 34, 191–218.

    Article  CAS  Google Scholar 

  3. Bjornsti, M.-A., Knab, A. M., and Benedetti, P. (1994) Yeast Saccharomyces cerevisiae as a model system to study the cytotoxic activity of the antitumor drug camptothecin. Cancer Chemother. Pharmacol. 34, S1–S5.

    Article  CAS  Google Scholar 

  4. Benedetti, P., Fiorani, P., Capuani, L., and Wang, J. C. (1993) Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine. Cancer Res. 53, 4343–4348.

    CAS  PubMed  Google Scholar 

  5. Bjornsti, M.-A., Benedetti, P., Viglianti, G. A., and Wang, J. C. (1989) 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.

    CAS  PubMed  Google Scholar 

  6. Bjornsti, M.-A. and Wang, J. C. (1987) Expression of Yeast DNA Topoisomerase I can Complement a Conditional-lethal DNA Topoisomerase I Mutation in Esherichia coli. Proc. Natl. Acad. Sci. USA 84, 8971–8975.

    Article  CAS  Google Scholar 

  7. Bjornsti, M.-A. (1991) DNA topoisomerases. Curr. Opin. Struct. Biol. 1, 99–103.

    Article  CAS  Google Scholar 

  8. Champoux, J. (1990) Mechanistic aspects of type-I topoisomerases, in DNA Topology and its Biological Effects (Cozzarelli, N. R. and Wang, J. C., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 217–242.

    Google Scholar 

  9. Christiansen, K., Svejstrup, A. B. D., Andersen, A. H., and Westergaard, O. (1993) Eukaryotic topoisomerase I-mediated cleavage requires bipartite DNA interaction. J. Biol. Chem. 268, 9690–9701.

    Article  CAS  Google Scholar 

  10. Eng, W.-K., Pandit, S. D., and Sternglanz, R. (1989) Mapping of the Active Site Tyrosine of Eukaryotic DNA Topoisomerase I. J. Biol. Chem. 264, 13,373–13,376.

    Article  CAS  Google Scholar 

  11. Gupta, M., Fujimori, A., and Pommier, Y. (1995) Eukaryotic DNA topoisomerase I. BBA 1262, 1–14.

    PubMed  Google Scholar 

  12. Knab, A. M., Fertala, J., and Bjornsti, M.-A. (1993) Mechanisms of camptothecin resistance in yeast DNA topoisomerase I mutants. J. Biol. Chem. 268, 22,322–22,330.

    Article  CAS  Google Scholar 

  13. Knab, A. M., Fertala, J., and Bjornsti, M.-A. (1995) A camptothecin-resistant DNA toposiomerase I mutant exhibits altered sensitivities to other DNA topoisomerase poisons. J. Biol. Chem. 270, 6141–6148.

    Article  CAS  Google Scholar 

  14. Kubota, N., Kanazawa, F., Nishio, K., Takeda, Y., Ohmori, T., Fujiwara, T., et al. (1992) Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line. Biochem. Biophys. Res. Comm. 188, 571–577.

    Article  CAS  Google Scholar 

  15. Levin, N. A., Bjornsti, M.-A., and Fink, G. R. (1993) A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest. Genetics 133, 799–814.

    Article  CAS  Google Scholar 

  16. Lue, N., Sharma, A., Mondragon, A., and Wang, J. C. (1995) A 26 kDa yeast DNA topoisomerase I fragment—crystallographic structure and mechanistic implications. Structure 3, 1315–1322.

    Article  CAS  Google Scholar 

  17. Pommier, Y. (1996) Eukaryotic DNA topoisomerase I—genome gatekeeper and its intruders, camptothecins. Semin. Oncol. 23, 3–10.

    CAS  PubMed  Google Scholar 

  18. Stewart, L., Ireton, G. C., and Champoux, J. J. (1996) The domain organization of human topoisomerase I. J. Biol. Chem. 271, 7602–7608.

    Article  CAS  Google Scholar 

  19. Stewart, L., Ireton, G. C., Parker, L. H., Madden, K. R., and Champoux, J. J. (1996) Biochemical and biophysical analysis of recombinant forms of human DNA topoisomerase I. J. Biol. Chem. 271, 7593–7601.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Holm, C., Goto, T., Wang, J. C., and Botstein, D. (1985) DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41, 553–563.

    Article  CAS  Google Scholar 

  22. Goto, T. and Wang, J. C. (1985) Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II. Proc. Natl. Acad. Sci. USA 82, 7178–7182.

    Article  CAS  Google Scholar 

  23. Castano, I. B., Heathpagliuso, S., Sadoff, B. U., Fitzhugh, D. J., and Christman, M. F. (1996) A novel family of Trf (DNA topoisomerase L-related function) genes required for proper nuclear segregation. Nucleic Acids Res. 24, 2404–2410.

    Article  CAS  Google Scholar 

  24. West, R. W., Yocum, R. R., and Ptashne, M. (1984) Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol. Cell. Biol. 4, 2467–2478.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lindsley, J. E. and Wang, J. C. (1991) Proteolysis patterns of epitopically labeled yeast DNA topoisomerase II suggest an allosteric transition in the enzyme induced by ATP binding. Proc. Natl. Acad. Sci. USA 88, 10,485–10,489.

    Article  CAS  Google Scholar 

  26. Kaiser, C., Michaelis, S., and Mitchell, A. (1994) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 234.

    Google Scholar 

  27. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 198.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Science, St. Jude— Children’s Research Hospital, Memphis, TN, USA

    Mary-Ann Bjornsti

  2. Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA, USA

    Jolanta Fertala

Authors
  1. Mary-Ann Bjornsti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jolanta Fertala
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. St. Jude Children’s Research Hospital, Memphis, TN, USA

    Mary-Ann Bjornsti

  2. Vanderbilt University School of Medicine, Nashville, TN, USA

    Neil Osheroff

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc.

About this protocol

Cite this protocol

Bjornsti, MA., Fertala, J. (1999). Overexpression and Purification of DNA Topoisomerase I from Yeast. In: Bjornsti, MA., Osheroff, N. (eds) DNA Topoisomerase Protocols. Methods in Molecular Biology, vol 94. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-259-7:179

Download citation

  • DOI: https://doi.org/10.1385/1-59259-259-7:179

  • Published:

  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-0-89603-444-0

  • Online ISBN: 978-1-59259-259-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation