Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Dec;11(12):6268–6278. doi: 10.1128/mcb.11.12.6268

Role of EBNA-1 in arresting replication forks at the Epstein-Barr virus oriP family of tandem repeats.

V Dhar 1, C L Schildkraut 1
PMCID: PMC361815  PMID: 1658629

Abstract

The 20-member family of 30-bp tandem repeats located within the oriP region of Epstein-Barr virus (EBV) can act as a transcriptional enhancer in the presence of EBV nuclear antigen 1 (EBNA-1). A replication fork barrier and a termination site of plasmid replication in human B cells is also found within or near the EBV tandem repeats. Within each tandem repeat is a consensus binding sequence for the EBNA-1 protein that is required for extrachromosomal maintenance of oriP-containing plasmids. To investigate the factors that contribute to the arrest of replication forks and termination in the region of the family of repeats, we have used an in vitro replication system in which replication of EBV recombinant plasmids is initiated from the simian virus 40 (SV40) DNA replication origin in the presence of SV40 T antigen and soluble extracts prepared from human cells. The system can support bidirectional replication, initiating from the SV40 DNA origin with termination occurring in a region opposite the origin. Using two-dimensional agarose gel electrophoresis, we observed a barrier to replication forks in the presence of EBNA-1 in the region of the EBV repeats. Termination occurs at or near the tandem repeats in a manner similar to that observed in vivo (T.A. Gahn and C.L. Schildkraut, Cell 58:527-535, 1989). Reducing the number of repeats from 20 to 6 had little effect on the strength of the replication fork barrier. In the absence of EBNA-1, replication forks also arrested at the EBV repeats, but at a much lower efficiency. The addition of competitor DNA containing the EBV family of repeats can almost completely abolish the replication barrier produced in the presence of EBNA-1.

Full text

PDF
6268

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams A. Replication of latent Epstein-Barr virus genomes in Raji cells. J Virol. 1987 May;61(5):1743–1746. doi: 10.1128/jvi.61.5.1743-1746.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arrand J. R., Rymo L. Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells. J Virol. 1982 Feb;41(2):376–389. doi: 10.1128/jvi.41.2.376-389.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  4. Bedinger P., Hochstrasser M., Jongeneel C. V., Alberts B. M. Properties of the T4 bacteriophage DNA replication apparatus: the T4 dda DNA helicase is required to pass a bound RNA polymerase molecule. Cell. 1983 Aug;34(1):115–123. doi: 10.1016/0092-8674(83)90141-1. [DOI] [PubMed] [Google Scholar]
  5. Bedinger P., Munn M., Alberts B. M. Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates. J Biol Chem. 1989 Oct 5;264(28):16880–16886. [PubMed] [Google Scholar]
  6. Bell L., Byers B. Separation of branched from linear DNA by two-dimensional gel electrophoresis. Anal Biochem. 1983 Apr 15;130(2):527–535. doi: 10.1016/0003-2697(83)90628-0. [DOI] [PubMed] [Google Scholar]
  7. Brewer B. J., Fangman W. L. A replication fork barrier at the 3' end of yeast ribosomal RNA genes. Cell. 1988 Nov 18;55(4):637–643. doi: 10.1016/0092-8674(88)90222-x. [DOI] [PubMed] [Google Scholar]
  8. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  9. Brewer B. J. When polymerases collide: replication and the transcriptional organization of the E. coli chromosome. Cell. 1988 Jun 3;53(5):679–686. doi: 10.1016/0092-8674(88)90086-4. [DOI] [PubMed] [Google Scholar]
  10. Chittenden T., Lupton S., Levine A. J. Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication. J Virol. 1989 Jul;63(7):3016–3025. doi: 10.1128/jvi.63.7.3016-3025.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DePamphilis M. L. Transcriptional elements as components of eukaryotic origins of DNA replication. Cell. 1988 Mar 11;52(5):635–638. doi: 10.1016/0092-8674(88)90398-4. [DOI] [PubMed] [Google Scholar]
  12. Deb S., DeLucia A. L., Baur C. P., Koff A., Tegtmeyer P. Domain structure of the simian virus 40 core origin of replication. Mol Cell Biol. 1986 May;6(5):1663–1670. doi: 10.1128/mcb.6.5.1663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Deuschle U., Hipskind R. A., Bujard H. RNA polymerase II transcription blocked by Escherichia coli lac repressor. Science. 1990 Apr 27;248(4954):480–483. doi: 10.1126/science.2158670. [DOI] [PubMed] [Google Scholar]
  14. Frappier L., O'Donnell M. Overproduction, purification, and characterization of EBNA1, the origin binding protein of Epstein-Barr virus. J Biol Chem. 1991 Apr 25;266(12):7819–7826. [PubMed] [Google Scholar]
  15. Gahn T. A., Schildkraut C. L. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell. 1989 Aug 11;58(3):527–535. doi: 10.1016/0092-8674(89)90433-9. [DOI] [PubMed] [Google Scholar]
  16. Hampar B., Tanaka A., Nonoyama M., Derge J. G. Replication of the resident repressed Epstein-Barr virus genome during the early S phase (S-1 period) of nonproducer Raji cells. Proc Natl Acad Sci U S A. 1974 Mar;71(3):631–633. doi: 10.1073/pnas.71.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang C. C., Hearst J. E. Pauses at positions of secondary structure during in vitro replication of single-stranded fd bacteriophage DNA by T4 DNA polymerase. Anal Biochem. 1980 Mar 15;103(1):127–139. doi: 10.1016/0003-2697(80)90246-8. [DOI] [PubMed] [Google Scholar]
  18. Khatri G. S., MacAllister T., Sista P. R., Bastia D. The replication terminator protein of E. coli is a DNA sequence-specific contra-helicase. Cell. 1989 Nov 17;59(4):667–674. doi: 10.1016/0092-8674(89)90012-3. [DOI] [PubMed] [Google Scholar]
  19. Kuempel P. L., Pelletier A. J., Hill T. M. Tus and the terminators: the arrest of replication in prokaryotes. Cell. 1989 Nov 17;59(4):581–583. doi: 10.1016/0092-8674(89)90001-9. [DOI] [PubMed] [Google Scholar]
  20. Kuhn A., Bartsch I., Grummt I. Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases. Nature. 1990 Apr 5;344(6266):559–562. doi: 10.1038/344559a0. [DOI] [PubMed] [Google Scholar]
  21. Lai C. J., Nathans D. Non-specific termination of simian virus 40 DNA replication. J Mol Biol. 1975 Sep 5;97(1):113–118. doi: 10.1016/s0022-2836(75)80026-x. [DOI] [PubMed] [Google Scholar]
  22. Lee E. H., Kornberg A., Hidaka M., Kobayashi T., Horiuchi T. Escherichia coli replication termination protein impedes the action of helicases. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9104–9108. doi: 10.1073/pnas.86.23.9104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Linskens M. H., Huberman J. A. Organization of replication of ribosomal DNA in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4927–4935. doi: 10.1128/mcb.8.11.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lupton S., Levine A. J. Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells. Mol Cell Biol. 1985 Oct;5(10):2533–2542. doi: 10.1128/mcb.5.10.2533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Martín-Parras L., Hernández P., Martínez-Robles M. L., Schvartzman J. B. Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis. J Mol Biol. 1991 Aug 20;220(4):843–853. doi: 10.1016/0022-2836(91)90357-c. [DOI] [PubMed] [Google Scholar]
  27. McKnight S. L., Bustin M., Miller O. L., Jr Electron microscopic analysis of chromosome metabolism in the Drosophila melanogaster embryo. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):741–754. doi: 10.1101/sqb.1978.042.01.075. [DOI] [PubMed] [Google Scholar]
  28. Rawlins D. R., Milman G., Hayward S. D., Hayward G. S. Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell. 1985 Oct;42(3):859–868. doi: 10.1016/0092-8674(85)90282-x. [DOI] [PubMed] [Google Scholar]
  29. Reisman D., Sugden B. trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1. Mol Cell Biol. 1986 Nov;6(11):3838–3846. doi: 10.1128/mcb.6.11.3838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reisman D., Yates J., Sugden B. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol. 1985 Aug;5(8):1822–1832. doi: 10.1128/mcb.5.8.1822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rosa M. D., Gottlieb E., Lerner M. R., Steitz J. A. Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII. Mol Cell Biol. 1981 Sep;1(9):785–796. doi: 10.1128/mcb.1.9.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tack L. C., Proctor G. N. Two major replicating simian virus 40 chromosome classes. Synchronous replication fork movement is associated with bound large T antigen during elongation. J Biol Chem. 1987 May 5;262(13):6339–6349. [PubMed] [Google Scholar]
  34. Vaughn J. P., Dijkwel P. A., Hamlin J. L. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell. 1990 Jun 15;61(6):1075–1087. doi: 10.1016/0092-8674(90)90071-l. [DOI] [PubMed] [Google Scholar]
  35. Wen L. T., Tanaka A., Nonoyama M. Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells. J Virol. 1989 Aug;63(8):3315–3322. doi: 10.1128/jvi.63.8.3315-3322.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wysokenski D. A., Yates J. L. Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus. J Virol. 1989 Jun;63(6):2657–2666. doi: 10.1128/jvi.63.6.2657-2666.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yates J., Warren N., Reisman D., Sugden B. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3806–3810. doi: 10.1073/pnas.81.12.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES