DNA Viruses pp 267-276 | Cite as

DNA Affinity Purification of Epstein-Barr Virus OriP-Binding Proteins

  • Constandache Atanasiu
  • Larissa Lezina
  • Paul M. Lieberman
Part of the Methods in Molecular Biology book series (MIMB, volume 292)


DNA affinity purification has been used to identify cellular and viral proteins associated with the Epstein-Barr virus origin of plasmid DNA replication. This approach allows for a one- or two-step purification scheme of high-affinity DNA binding proteins from crude nuclear extracts. Additionally, this approach may be useful for isolation of proteins that are found in the insoluble fractions of the nuclear matrix or scaffold.

Key Words

DNA affinity magnetic beads OriP EBNA-1 telomere repeat binding factors 


  1. 1.
    Kadonaga, J. T. and Tjian, R. (1986) Affinity purification of sequence-specific DNA binding proteins. Proc. Natl. Acad. Sci. USA 83, 5889–5893.PubMedCrossRefGoogle Scholar
  2. 2.
    Rosenfeld, P. J. and Kelly, T. J. (1986) Purification of nuclear factor I by DNA recognition site affinity chromatography. J. Biol. Chem. 261, 1398–1408.PubMedGoogle Scholar
  3. 3.
    Gadgil, H., Oak, S. A., and Jarrett, H. W. (2001) Affinity purification of DNA-binding proteins. J. Biochem. Biophys. Methods 49, 607–624.PubMedCrossRefGoogle Scholar
  4. 4.
    Kadonaga, J. T. (1991) Purification of sequence-specific binding proteins by DNA affinity chromatography. Methods Enzymol. 208, 10–23.PubMedCrossRefGoogle Scholar
  5. 5.
    Ranish, J. A., Yudkovsky, N., and Hahn, S. (1999) Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13, 49–63.PubMedCrossRefGoogle Scholar
  6. 6.
    Kumar, R., Reynolds, D. M., Shevchenko, A., Shevchenko, A., Gladstone, S. D., Dalton, S. (2000) Forkhead transcription factors, Fkh1p and Fkh2p, collaborate with Mcm1p to control transcription required for M-phase. Curr. Biol. 10, 896–906.PubMedCrossRefGoogle Scholar
  7. 7.
    Sugden, B. and Leight, E. R. (2001) Molecular mechanisms of maintenance and disruption of virus latency, in Epstein-Barr Virus and Human Cancer (Takada, K., ed.), Springer, Heidelberg, pp. 3–11.Google Scholar
  8. 8.
    Leight, E. R. and Sugden, B. (2000) EBNA-1: a protein pivotal to latent infection by Epstein-Barr virus. Rev. Med. Virol. 10, 83–100.PubMedCrossRefGoogle Scholar
  9. 9.
    Yates, J. L. Warren, N., Reisman, D., Sugden, B., et al. (1984) A cis-acting element from Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc. Natl. Acad. Sci. USA 81, 3806–3810.PubMedCrossRefGoogle Scholar
  10. 10.
    Rawlins, D. R. Millman, G., Hayward, S. D., Howard, G. S. (1985) Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell 42, 859–868.PubMedCrossRefGoogle Scholar
  11. 11.
    Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Constandache Atanasiu
    • 1
  • Larissa Lezina
    • 2
  • Paul M. Lieberman
    • 2
  1. 1.The Wistar InstitutePhiladelphia
  2. 2.The Wistar InstitutePhiladelphia

Personalised recommendations