Skip to main content

Binding of DNA Topoisomerases I and II to Replication Origins

  • Protocol
  • First Online:

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

Abstract

The interaction of DNA topology modifying enzymes with eukaryotic DNA replication origins can be detected with nucleotide precision exploiting the action of enzyme poisons specific for type I or type II DNA topoisomerases. Using the topoisomerase I poison camptothecin and the topoisomerase II poison VP16, the precise sites of interaction of these enzymes around the lamin B2 origin have been identified at different points in the cell cycle. The procedure can be applied to any origin for which the sequence has been identified within approximately 1 kb.

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

Buying options

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   149.00
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Kornberg, A. (1984) Enzyme studies on the replication of the Escherichia coli chromosome. Adv Exp Med Biol 179, 3–16.

    PubMed  CAS  Google Scholar 

  2. Halmer, L., Vestner, B. and Gruss, C. (1998) Involvement of topoisomerases in the initiation of simian virus 40 minichromosome replication. J Biol Chem 273, 34792–34798.

    Article  PubMed  CAS  Google Scholar 

  3. Hu, Y., Clower, R. V. and Melendy, T. (2006) Cellular topoisomerase I modulates origin binding by bovine papilloma virus type 1 E1. J Virol 80, 4363–4371.

    Article  PubMed  CAS  Google Scholar 

  4. Kawanishi, M. (1993) Topoisomerase I and II activities are required for Epstein-Barr virus replication. J Gen Virol 74, 2263–2268.

    Article  PubMed  CAS  Google Scholar 

  5. Mitkova, A.V., Biswas-Fiss, E. E. and Biswas, S.B. (2005) Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex. J Biol Chem 280, 6285–6292.

    Article  PubMed  CAS  Google Scholar 

  6. Remus, D., Beall, E.L. and Botchan, M.R. (2004) DNA topology, not DNA sequence, is a critical determinant for Drosophila. ORC-DNA binding. EMBO J 23, 897–907.

    Article  PubMed  CAS  Google Scholar 

  7. Abdurashidova, G., Radulescu, S., Sandoval, O., Zahariev S., Danailov, M. B., Demidovich, A., Santamaria, L., Biamonti, G., Riva, S. and Falaschi, A. (2007) Functional interactions of DNA topoisomerases with a human replication origin. EMBO J 26, 998–1009.

    Article  PubMed  CAS  Google Scholar 

  8. Pommier, Y., Pourquier, P., Fan, Y. and Strumberg, D. (1998) Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim Biophys Acta 1400, 83–105.

    Article  PubMed  CAS  Google Scholar 

  9. Burden, D.A. and Osheroff, N. (1998). Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim Biophys Acta 1400, 139–154.

    Article  PubMed  CAS  Google Scholar 

  10. Strumberg, D., Pilon, A.A., Smith, M., Hickey, R., Malkas, L. and Pommier, Y. (2000) 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.

    Article  PubMed  CAS  Google Scholar 

  11. 11.Mueller, P.A., Wold, B. and Garrity, P.A. (2001) Ligation-mediated PCR for genomic sequencing and footprinting. In Current protocols in molecular biology (New York: John Wiley & Sons, Inc.), pp. 15.3.1–15.3.26.

    Google Scholar 

  12. Komura, J. and Riggs, A.D. (1998) Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts. Nucleic Acids Res 26, 1807–1811.

    Article  PubMed  CAS  Google Scholar 

  13. Quivy, J. P. and Becker P.B. (1993) An improved protocol for genomic sequencing and footprinting by ligation-mediated PCR. Nucleic Acids Res 21, 2779–2781.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Falaschi, A. (2009). Binding of DNA Topoisomerases I and II to Replication Origins. In: Clarke, D. (eds) DNA Topoisomerases. Methods in Molecular Biology™, vol 582. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-340-4_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-340-4_11

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-339-8

  • Online ISBN: 978-1-60761-340-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics