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Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres

Nature Genetics volume 25, pages 347–352 (2000)Cite this article

Abstract

Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1–4), which has been proposed to remodel telomeres into large duplex loops5 (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to γ-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.

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Figure 1: Association of TRF2 with the MRE11 complex.
Figure 4: γ-irradiation does not affect the telomeric localization of TRF2 or its interaction with the MRE11 complex.
Figure 2: Presence of RAD50, MRE11 and NBS1 at telomeres.
Figure 3: Cell-cycle-regulated association of NBS1 with TRF2 and telomeres.

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References

  1. Broccoli, D., Smogorzewska, A., Chong, L. & de Lange, T. Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nature Genet. 17, 231–235 (1997).

    Article  CAS  Google Scholar 

  2. Bilaud, T. et al. Telomeric localization of TRF2, a novel human telobox protein. Nature Genet. 17, 236–239 (1997).

    Article  CAS  Google Scholar 

  3. van Steensel, B., Smogorzewska, A. & de Lange, T. TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413 (1998).

    Article  CAS  Google Scholar 

  4. Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & de Lange, T. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283, 1321–1325 (1999).

    Article  CAS  Google Scholar 

  5. Griffith, J.D. et al. Mammalian telomeres end in a large duplex loop. Cell 97, 503–514 (1999).

    Article  CAS  Google Scholar 

  6. Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996).

    Article  CAS  Google Scholar 

  7. Li, B., Oestreich, S. & de Lange, T. Identification of human Rap1: implications for telomere evolution. Cell 101, 471–483 (2000).

    Article  CAS  Google Scholar 

  8. Dolganov, G.M. et al. Human Rad50 is physically associated with hMre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol. Cell. Biol. 16, 4832–4841 (1996).

    Article  CAS  Google Scholar 

  9. Carney, J.P. et al. The hMre11/hRad50 protein complex and Nijmegen Breakage Syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93, 477–486 (1998).

    Article  CAS  Google Scholar 

  10. Haber, J.E. The many interfaces of Mre11. Cell 95, 583–586 (1998).

    Article  CAS  Google Scholar 

  11. Nelms, B.E., Maser, R.S., MacKay, J.F., Lagally, M.G. & Petrini, J.H.J. In situ visualization of DNA double-strand break repair in human fibroblasts. Science 280, 590–592 (1998).

    Article  CAS  Google Scholar 

  12. Lim, D.-S. et al. ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature 404, 613–617 (2000).

    Article  CAS  Google Scholar 

  13. Bressan, D.A., Baxter, B.K. & Petrini, J.H.J. The Mre11/Rad50/Xrs2 protein complex facilitates homologous recombination-based double strand break repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 7681–7687 (1999).

    Article  CAS  Google Scholar 

  14. Usui, T., Ohta, T., Oshiumi, J., Ogawa, T.H. & Ogawa, T. Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95, 705–716 (1998).

    Article  CAS  Google Scholar 

  15. Nugent, C. et al. Telomere maintenance is dependent on activities required for end repair of double-strand breaks. Curr. Biol. 8, 657–660 (1998).

    Article  CAS  Google Scholar 

  16. Boulton, S.J. & Jackson, S.P. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 17, 1819–1828 (1998).

    Article  CAS  Google Scholar 

  17. Le, S., Moore, J.K., Haber, J.E. & Greider, C.W. RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics 152, 143–152 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Xiao, Y. & Weaver, D.T. Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. Nucleic Acids Res. 25, 2985–2991 (1997).

    Article  CAS  Google Scholar 

  19. Luo, G. et al. Disruption of mRad50 causes ES cell lethality, abnormal embryonic development and sensitivity to ionizing radiation. Proc. Natl Acad. Sci. USA 96, 7376–7381 (1999).

    Article  CAS  Google Scholar 

  20. van Steensel, B. & de Lange, T. Control of telomere length by the human telomeric protein TRF1. Nature 385, 740–743 (1997).

    Article  CAS  Google Scholar 

  21. Chong, L. et al. A human telomeric protein. Science 270, 1663–1667 (1995).

    Article  CAS  Google Scholar 

  22. Maser, R.S., Monsen, K.J., Nelms, B. & Petrini, J.H.J. hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol. Cell. Biol. 17, 6087–6096 (1997).

    Article  CAS  Google Scholar 

  23. Bryan, T.M., Englezou, A., Gupta, J., Bacchetti, S. & Reddel, R.R. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J. 14, 4240–4248 (1995).

    Article  CAS  Google Scholar 

  24. Yeager, T.R. et al. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res. 59, 4175–4179 (1999).

    CAS  PubMed  Google Scholar 

  25. Stewart, G. et al. The DNA double strand break repair gene hMre11, is mutated in individuals with a new ataxia telangiectasia like disorders (ATLD). Cell 99, 577–587 (1999).

    Article  CAS  Google Scholar 

  26. Martin, S.G., Laroche, T., Suka, N., Grunstein, M. & Gasser, S.M. Relocalization of telomeric Ku and Sir proteins in response to DNA strand breaks in yeast. Cell 97, 621–633 (1999).

    Article  CAS  Google Scholar 

  27. Mills, K.D., Sinclair, D.A. & Guarente, L. Mec1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks. Cell 97, 609–620 (1999).

    Article  CAS  Google Scholar 

  28. Moore, J.K. & Haber, J.E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2164–2173 (1996).

    Article  CAS  Google Scholar 

  29. Paull, T.T. & Gellert, M. Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. Genes Dev. 13, 1276–1288 (1999).

    Article  CAS  Google Scholar 

  30. Harley, C.B. Telomeres and aging. in Telomeres (eds Blackburn, E.H. & Greider, C.W.) 247–265 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 1995).

    Google Scholar 

  31. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  Google Scholar 

  32. Shevchenko, A. et al. Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024 (1997).

    Article  CAS  Google Scholar 

  33. Mann, M. & Wilm, M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank N. Heintz and W. Lee for HeLa nuclear extract; J. Karlseder for help with FACS analysis, pCDNA3-TRF2ΔBΔM and comments on the manuscript; A. Smogorzewska for HeLa cells infected with TRF2ΔBΔM retrovirus and comments on the manuscript; and B. Li, G. Celli, S. Smith and J. Ye for discussion. X.-D.Z. is supported by a Canadian MRC postdoctoral fellowship. B.K. was supported (in part) by a long-term post-doctoral fellowship from the EMBO. The laboratory of M.M. at the University of Southern Denmark is supported by a grant from the Danish National Research Foundation to the Center of Experimental BioInformatics (CEBI). J.H.J.P. is supported by NIH/NCI GM56888, GM59413 and the Milwaukee Foundation. This work was supported by a grant from the NIH (GM49046) to T.d.L.

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Authors and Affiliations

  1. The Rockefeller University, New York, New York, USA

    Xu-Dong Zhu & Titia de Lange

  2. Protein Interaction Laboratory, University of Southern Denmark, Odense, Denmark

    Bernhard Küster & Matthias Mann

  3. Laboratory of Genetics, University of Wisconsin Medical School, Madison, Wisconsin, USA

    John H.J. Petrini

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Correspondence to Titia de Lange.

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Zhu, XD., Küster, B., Mann, M. et al. Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet 25, 347–352 (2000). https://doi.org/10.1038/77139

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