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The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity

Abstract

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase1,2,3. Shelterin includes the heterodimeric POT1–TPP1 protein, which binds the telomeric single-stranded DNA tail4,5,6,7,8,9. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event)9,10,11,12,13,14. Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP1 (refs 15, 16, 17). Here we identify separation-of-function mutants of human TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding human telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anticancer drug development.

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Figure 1: Separation-of-function mutants of TPP1 affect telomerase processivity without affecting telomere complex formation.
Figure 2: TPP1 mutations that disrupt telomerase stimulation also disrupt telomerase binding.
Figure 3: TPP1 TEL-patch mutants fail to stimulate telomere lengthening in human cells.
Figure 4: Failure to stimulate telomere lengthening correlates with inability to recruit telomerase to telomeres.

References

  1. Palm, W. & de Lange, T. How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42, 301–334 (2008)

    CAS  Article  Google Scholar 

  2. Greider, C. W. & Blackburn, E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331–337 (1989)

    ADS  CAS  Article  Google Scholar 

  3. Lingner, J. et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276, 561–567 (1997)

    CAS  Article  Google Scholar 

  4. Baumann, P. & Cech, T. R. Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171–1175 (2001)

    ADS  CAS  Article  Google Scholar 

  5. Houghtaling, B. R., Cuttonaro, L., Chang, W. & Smith, S. A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2. Curr. Biol. 14, 1621–1631 (2004)

    CAS  Article  Google Scholar 

  6. Liu, D. et al. PTOP interacts with POT1 and regulates its localization to telomeres. Nature Cell Biol. 6, 673–680 (2004)

    CAS  Article  Google Scholar 

  7. Ye, J. Z. et al. POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev. 18, 1649–1654 (2004)

    CAS  Article  Google Scholar 

  8. Lei, M., Podell, E. R. & Cech, T. R. Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection. Nature Struct. Mol. Biol. 11, 1223–1229 (2004)

    CAS  Article  Google Scholar 

  9. Wang, F. et al. The POT1–TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506–510 (2007)

    ADS  CAS  Article  Google Scholar 

  10. Lue, N. F. Adding to the ends: what makes telomerase processive and how important is it? Bioessays 26, 955–962 (2004)

    CAS  Article  Google Scholar 

  11. Latrick, C. M. & Cech, T. R. POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J. 29, 924–933 (2010)

    CAS  Article  Google Scholar 

  12. Tejera, A. M. et al. TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice. Dev. Cell 18, 775–789 (2010)

    CAS  Article  Google Scholar 

  13. Abreu, E. et al. TIN2-tethered TPP1 recruits human telomerase to telomeres in vivo. Mol. Cell. Biol. 30, 2971–2982 (2010)

    CAS  Article  Google Scholar 

  14. Xin, H. et al. TPP1 is a homologue of ciliate TEBP-β and interacts with POT1 to recruit telomerase. Nature 445, 559–562 (2007)

    ADS  CAS  Article  Google Scholar 

  15. Denchi, E. L. & de Lange, T. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature 448, 1068–1071 (2007)

    ADS  CAS  Article  Google Scholar 

  16. Guo, X. et al. Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis. EMBO J. 26, 4709–4719 (2007)

    CAS  Article  Google Scholar 

  17. Hockemeyer, D. et al. Telomere protection by mammalian Pot1 requires interaction with Tpp1. Nature Struct. Mol. Biol. 14, 754–761 (2007)

    CAS  Article  Google Scholar 

  18. Cristofari, G. & Lingner, J. Telomere length homeostasis requires that telomerase levels are limiting. EMBO J. 25, 565–574 (2006)

    CAS  Article  Google Scholar 

  19. Zaug, A. J., Podell, E. R., Nandakumar, J. & Cech, T. R. Functional interaction between telomere protein TPP1 and telomerase. Genes Dev. 24, 613–622 (2010)

    CAS  Article  Google Scholar 

  20. Zhong, F. L. et al. TPP1 OB-fold domain controls telomere maintenance by recruiting telomerase to chromosome ends. Cell 150, 481–494 (2012)

    CAS  Article  Google Scholar 

  21. Sexton, A. N., Youmans, D. T. & Collins, K. Specificity requirements for human telomere protein interaction with telomerase holoenzyme. J. Biol. Chem.. http://dx.doi:10.1074/jbc.M112.394767jbc.M112.394767 (2012)

  22. Miyoshi, T., Kanoh, J., Saito, M. & Ishikawa, F. Fission yeast Pot1-Tpp1 protects telomeres and regulates telomere length. Science 320, 1341–1344 (2008)

    ADS  CAS  Article  Google Scholar 

  23. Moser, B. A., Chang, Y. T., Kosti, J. & Nakamura, T. M. Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast. Nature Struct. Mol. Biol. 18, 1408–1413 (2011)

    CAS  Article  Google Scholar 

  24. Yamazaki, H., Tarumoto, Y. & Ishikawa, F. Tel1ATM and Rad3ATR phosphorylate the telomere protein Ccq1 to recruit telomerase and elongate telomeres in fission yeast. Genes Dev. 26, 241–246 (2012)

    CAS  Article  Google Scholar 

  25. Park, J. I. et al. Telomerase modulates Wnt signalling by association with target gene chromatin. Nature 460, 66–72 (2009)

    ADS  CAS  Article  Google Scholar 

  26. Majerská, J., Sykorova, E. & Fajkus, J. Non-telomeric activities of telomerase. Mol. Biosyst. 7, 1013–1023 (2011)

    Article  Google Scholar 

  27. Abreu, E., Terns, R. M. & Terns, M. P. Visualization of human telomerase localization by fluorescence microscopy techniques. Methods Mol. Biol. 735, 125–137 (2011)

    CAS  Article  Google Scholar 

  28. Weidenfeld, I. et al. Inducible expression of coding and inhibitory RNAs from retargetable genomic loci. Nucleic Acids Res. 37, e50 (2009)

    Article  Google Scholar 

  29. Abell, A. N. et al. Rac2D57N, a dominant inhibitory Rac2 mutant that inhibits p38 kinase signaling and prevents surface ruffling in bone-marrow-derived macrophages. J. Cell Sci. 117, 243–255 (2004)

    CAS  Article  Google Scholar 

  30. Berger, S. M. et al. Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs. Nucleic Acids Res. 38, e168 (2010)

    Article  Google Scholar 

  31. Mossessova, E. & Lima, C. D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000)

    CAS  Article  Google Scholar 

  32. Nandakumar, J., Podell, E. R. & Cech, T. R. How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA. Proc. Natl Acad. Sci. USA 107, 651–656 (2010)

    ADS  CAS  Article  Google Scholar 

  33. Weidenfeld, I. Inducible microRNA-mediated knockdown of the endogenous human lamin A/C gene. Methods Mol. Biol. 815, 289–305 (2012)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank T. de Lange, M. Terns and S. Langer for suggestions and sharing protocols; T. Nahreini for maintenance of the departmental tissue culture facility; J. Friedman and G. Voeltz for help with confocal microscopy; and A. Berman, S. Borah and M. Nakashima for critical reading of the manuscript. T.R.C. is an investigator of the Howard Hughes Medical Institute (HHMI). J.N. was an HHMI fellow of the Helen Hay Whitney Foundation during a major part of this study and is supported by the National Cancer Institute of the National Institutes of Health under award number K99CA167644. This work was supported in part by US National Institutes of Health grant R01GM29090 to L.A.L. and R01GM099705 to T.R.C.

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Authors and Affiliations

Authors

Contributions

J.N. and T.R.C. conceived the project and designed experiments with help from I.W. and L.A.L. on biological aspects. C.F.B. with help from J.N. and A.J.Z. conducted protein purifications, DNA-binding assays and telomerase assays. J.N. and I.W. constructed the stable HeLa cell lines. J.N. performed all remaining experiments including molecular cloning, cell culture, co-IP, TRF analysis and IF–FISH. J.N. and T.R.C. wrote the paper.

Corresponding author

Correspondence to Thomas R. Cech.

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Competing interests

The authors declare competing financial interests: T.R.C., J.N., C.F.B. and I.W. have filed a patent application relating to the identification of the TEL patch of TPP1.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13, a Supplementary Discussion and additional references. Supplementary Figures 2-6 show in vitro DNA-binding and direct telomerase assays with TPP1-OB mutants, Supplementary Figure 7 shows an investigation of telomerase components contributing to TPP1-OB binding, and Supplementary Figures 8-13 show the development, validation, and utilization of the HeLa-based system used to address the biological phenotypes (chromosome end protection, telomere maintenance, and telomerase recruitment) of the TEL patch mutants of TPP1. The Supplementary Discussion describes how telomerase recruitment in budding yeast compares to that in humans and S. pombe. (PDF 9289 kb)

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Nandakumar, J., Bell, C., Weidenfeld, I. et al. The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity. Nature 492, 285–289 (2012). https://doi.org/10.1038/nature11648

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