Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo

Nature Structural & Molecular Biology volume 12, pages 847–854 (2005)Cite this article

Abstract

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPα and TEBPβ. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPα in attaching telomeres in the nucleus and in recruiting TEBPβ to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPβ in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle–dependent phosphorylation of TEBPβ.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of a macronuclear nanochromosome.
Figure 2: TEBPα attaches telomeres to a subnuclear structure and recruits TEBPβ to the attachment sites.
Figure 3: Silencing of TEBPα and TEBPβ gene expression by RNAi.
Figure 4: Folding of telomeric G-overhangs into an antiparallel G-quadruplex DNA structure requires both TEBPα and TEBPβ in vivo.
Figure 5: TEBPβ requires TEBPα in order to interact with telomeric DNA in vitro.
Figure 6: Both TEBPs are required for G-quadruplex DNA formation and the basic C terminus of TEBPβ is essential.
Figure 7: Phosphorylated TEBPβ does not interact with telomeric DNA bound by TEBPα in vitro.
Figure 8: TEBPβ is phosphorylated during S phase, and inhibition of phosphorylation prevents the G-quadruplex DNA structure from being resolved during replication.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Zakian, V.A. Telomeres: beginning to understand the end. Science 270, 1601–1607 (1995).

    Article  CAS  Google Scholar 

  2. De Lange, T. Protection of mammalian telomeres. Oncogene 21, 532–540 (2002).

    Article  CAS  Google Scholar 

  3. Jonsson, F. & Lipps, H.J. The biology of telomeres in hypotrichous ciliates. in Telomerases, Telomeres and Cancer (eds. Krupp, G. & Parwaresch, R.) 205–222 (Landes Bioscience, Kluwer Academic/Plenum Publishers, Georgetown, Texas, USA, 2002).

    Google Scholar 

  4. Klobutcher, L.A., Swanton, M.T., Donini, P. & Prescott, D.M. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3′ terminus. Proc. Natl. Acad. Sci. USA 78, 3015–3019 (1981).

    Article  CAS  Google Scholar 

  5. Wellinger, R.J., Ethier, K., Labrecque, P. & Zakian, V.A. Evidence for a new step in telomere maintenance. Cell 85, 423–433 (1996).

    Article  CAS  Google Scholar 

  6. Makarov, V.L., Hirose, Y. & Langmore, J.P. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88, 657–666 (1997).

    Article  CAS  Google Scholar 

  7. Murzin, A.G. OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J. 12, 861–867 (1993).

    Article  CAS  Google Scholar 

  8. Mitton-Fry, R.M., Anderson, E.M., Hughes, T.R., Lundblad, V. & Wuttke, D.S. Conserved structure for single-stranded telomeric DNA recognition. Science 296, 145–147 (2002).

    Article  CAS  Google Scholar 

  9. Theobald, D.L. & Wuttke, D.S. Prediction of multiple tandem OB-fold domains in telomere end-binding proteins Pot1 and Cdc13. Structure (Camb) 12, 1877–1879 (2004).

    Article  CAS  Google Scholar 

  10. Gottschling, D.E. & Zakian, V.A. Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell 47, 195–205 (1986).

    Article  CAS  Google Scholar 

  11. Peersen, O.B., Ruggles, J.A. & Schultz, S.C. Dimeric structure of the Oxytricha nova telomere end-binding protein alpha-subunit bound to ssDNA. Nat. Struct. Biol. 9, 182–187 (2002).

    CAS  PubMed  Google Scholar 

  12. Gray, J.T., Celander, D.W., Price, C.M. & Cech, T.R. Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex. Cell 67, 807–814 (1991).

    Article  CAS  Google Scholar 

  13. Fang, G. & Cech, T.R. Oxytricha telomere-binding protein: DNA-dependent dimerization of the alpha and beta subunits. Proc. Natl. Acad. Sci. USA 90, 6056–6060 (1993).

    Article  CAS  Google Scholar 

  14. Horvath, M.P., Schweiker, V.L., Bevilacqua, J.M., Ruggles, J.A. & Schultz, S.C. Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963–974 (1998).

    Article  CAS  Google Scholar 

  15. Fang, G. & Cech, T.R. The beta subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA. Cell 74, 875–885 (1993).

    Article  CAS  Google Scholar 

  16. Sundquist, W.I. & Klug, A. Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops. Nature 342, 825–829 (1989).

    Article  CAS  Google Scholar 

  17. Williamson, J.R., Raghuraman, M.K. & Cech, T.R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell 59, 871–880 (1989).

    Article  CAS  Google Scholar 

  18. Lipps, H.J. In vitro aggregation of the gene-sized DNA molecules of the ciliate Stylonychia mytilus. Proc. Natl. Acad. Sci. USA 77, 4104–4107 (1980).

    Article  CAS  Google Scholar 

  19. Giraldo, R. & Rhodes, D. The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA. EMBO J. 13, 2411–2420 (1994).

    Article  CAS  Google Scholar 

  20. Zahler, A.M., Williamson, J.R., Cech, T.R. & Prescott, D.M. Inhibition of telomerase by G-quartet DNA structures. Nature 350, 718–720 (1991).

    Article  CAS  Google Scholar 

  21. Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. USA 98, 8572–8577 (2001).

    Article  CAS  Google Scholar 

  22. Meyer, G.F. & Lipps, H.J. The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus. Chromosoma 82, 309–314 (1981).

    Article  CAS  Google Scholar 

  23. Murti, K.G. & Prescott, D.M. Topological organization of DNA molecules in the macronucleus of hypotrichous ciliated protozoa. Chromosome Res. 10, 165–173 (2002).

    Article  CAS  Google Scholar 

  24. Lipps, H.J., Gruissem, W. & Prescott, D.M. Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova. Proc. Natl. Acad. Sci. USA 79, 2495–2499 (1982).

    Article  CAS  Google Scholar 

  25. Postberg, J. et al. Association of the telomere-telomere binding protein-complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication. J. Cell Sci. 114, 1861–1866 (2001).

    CAS  PubMed  Google Scholar 

  26. de Lange, T. Human telomeres are attached to the nuclear matrix. EMBO J. 11, 717–724 (1992).

    Article  CAS  Google Scholar 

  27. de Lara, J., Wydner, K.L., Hyland, K.M. & Ward, W.S. Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures. J. Cell. Biochem. 53, 213–221 (1993).

    Article  CAS  Google Scholar 

  28. Laroche, T., Martin, S.G., Tsai-Pflugfelder, M. & Gasser, S.M. The dynamics of yeast telomeres and silencing proteins through the cell cycle. J. Struct. Biol. 129, 159–174 (2000).

    Article  CAS  Google Scholar 

  29. Jonsson, F., Postberg, J., Schaffitzel, C. & Lipps, H.J. Organization of the macronuclear gene-sized pieces of stichotrichous ciliates into a higher order structure via telomere-matrix interactions. Chromosome Res. 10, 445–453 (2002).

    Article  Google Scholar 

  30. Jackson, D.A., Yuan, J. & Cook, P.R. A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. J. Cell Sci. 90, 365–378 (1988).

    CAS  PubMed  Google Scholar 

  31. Paschka, A.G. et al. The use of RNAi to analyze gene function in spirotrichous ciliates. Eur. J. Protistol. 39, 449–454 (2003).

    Article  Google Scholar 

  32. Schaffitzel, C., Hanes, J., Jermutus, L. & Pluckthun, A. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J. Immunol. Methods 231, 119–135 (1999).

    Article  CAS  Google Scholar 

  33. Lipps, H.J. et al. Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate Stylonychia mytilus. Cell 32, 435–441 (1983).

    Article  CAS  Google Scholar 

  34. Fang, G., Gray, J.T. & Cech, T.R. Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit. Genes Dev. 7, 870–882 (1993).

    Article  CAS  Google Scholar 

  35. Fang, G. & Cech, T.R. Characterization of a G-quartet formation reaction promoted by the beta- subunit of the Oxytricha telomere-binding protein. Biochemistry 32, 11646–11657 (1993).

    Article  CAS  Google Scholar 

  36. Horvath, M.P. & Schultz, S.C. DNA G-quartets in a 1.86 A resolution structure of an Oxytricha nova telomeric protein-DNA complex. J. Mol. Biol. 310, 367–377 (2001).

    Article  CAS  Google Scholar 

  37. Froelich-Ammon, S.J., Dickinson, B.A., Bevilacqua, J.M., Schultz, S.C. & Cech, T.R. Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha. Genes Dev. 12, 1504–1514 (1998).

    Article  CAS  Google Scholar 

  38. Hicke, B. et al. Phosphorylation of the Oxytricha telomere protein: possible cell cycle regulation. Nucleic Acids Res. 23, 1887–1893 (1995).

    Article  CAS  Google Scholar 

  39. Obenauer, J.C., Cantley, L.C. & Yaffe, M.B. Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31, 3635–3641 (2003).

    Article  CAS  Google Scholar 

  40. Juranek, S.A., Jönsson, F., Maercker, C. & Lipps, H.J. The telomeres of replicating macronuclear DNA molecules of the ciliate Stylonychia lemnae. Protistology 1, 148–151 (2000).

    Google Scholar 

  41. Kitagawa, M. et al. Butyrolactone I, a selective inhibitor of cdk2 and cdc2 kinase. Oncogene 8, 2425–2432 (1993).

    CAS  PubMed  Google Scholar 

  42. Rhodes, D. & Giraldo, R. Telomere structure and function. Curr. Opin. Struct. Biol. 5, 311–322 (1995).

    Article  CAS  Google Scholar 

  43. Riou, J.F. et al. Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands. Proc. Natl. Acad. Sci. USA 99, 2672–2677 (2002).

    Article  CAS  Google Scholar 

  44. Rezler, E.M., Bearss, D.J. & Hurley, L.H. Telomeres and telomerases as drug targets. Curr. Opin. Pharmacol. 2, 415–423 (2002).

    Article  CAS  Google Scholar 

  45. Ding, H. et al. Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein. Cell 117, 873–886 (2004).

    Article  CAS  Google Scholar 

  46. Ammermann, D., Steinbruck, G., von Berger, L. & Hennig, W. The development of the macronucleus in the ciliated protozoan Stylonychia mytilus. Chromosoma 45, 401–429 (1974).

    Article  CAS  Google Scholar 

  47. Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).

    Article  CAS  Google Scholar 

  48. Fairall, L., Chapman, L., Moss, H., de Lange, T. & Rhodes, D. Structure of the TRFH dimerization domain of the human telomeric proteins TRF and TRF2. Mol. Cell 8, 351–361 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.J.L., a European Molecular Biology Organization short-term fellowship to K.P. and a Wenner-Gren Foundations fellowship to T.S. We thank T. Cech (University of Colorado, Boulder, Colorado, USA) for providing antibodies against the TEBP subunits and C. Schaffitzel (Swiss Federal Institute of Technology, Zurich) for providing antibodies against G-quadruplex DNA structures.

Author information

Author notes

  1. Katrin Paeschke and Tomas Simonsson: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Cell Biology, University Witten/Herdecke, Stockumer Strasse 10, Witten, 58453, Germany

    Katrin Paeschke, Jan Postberg & Hans Joachim Lipps

  2. Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Tomas Simonsson & Daniela Rhodes

Authors
  1. Katrin Paeschke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomas Simonsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jan Postberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniela Rhodes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans Joachim Lipps
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Daniela Rhodes or Hans Joachim Lipps.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

A ClustalW sequence alignment of TEBPβ from Stylonychia lemnae and Oxytricha nova. (PDF 109 kb)

Supplementary Table 1

Stability of telomeric DNA upon RNAi mediated silencing of TEBPα or TEBPβ expression: no RNAi. (PDF 16 kb)

Supplementary Table 2

Stability of telomeric DNA upon RNAi mediated silencing of TEBPa or TEBPb expression: TEBPα RNAi. (PDF 16 kb)

Supplementary Table 3

Stability of telomeric DNA upon RNAi mediated silencing of TEBPa or TEBPb expression: TEBPβ RNAi. (PDF 16 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paeschke, K., Simonsson, T., Postberg, J. et al. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat Struct Mol Biol 12, 847–854 (2005). https://doi.org/10.1038/nsmb982

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb982

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing