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Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA

Nature Structural & Molecular Biology volume 17, pages 513–518 (2010)Cite this article

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Abstract

Telomerase is a specialized DNA polymerase that extends the 3′ ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B′ position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT–RNA template and TERT–telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

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Figure 1: Structure of the T. castaneum TERT–nucleic acid complex.
Figure 2: TERT–RNA template associations.
Figure 3: TERT–telomeric DNA associations.
Figure 4: DNA interactions with the primer grip region and the active site.
Figure 5: TERT rigid conformational changes upon nucleic acid binding.
Figure 6: Structural comparison of the RNA-DNA–bound TERT and HIV RT.

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References

  1. Gillis, A.J., Schuller, A.P. & Skordalakes, E. Structure of the Tribolium castaneum telomerase catalytic subunit TERT. Nature 455, 633–637 (2008).

    Article  CAS  Google Scholar 

  2. Greider, C.W. & Blackburn, E.H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405–413 (1985).

    Article  CAS  Google Scholar 

  3. Harley, C.B., Futcher, A.B. & Greider, C.W. Telomeres shorten during ageing of human fibroblasts. Nature 345, 458–460 (1990).

    Article  CAS  Google Scholar 

  4. Kim, N.W. et al. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2015 (1994).

    Article  CAS  Google Scholar 

  5. Harley, C.B., Vaziri, H., Counter, C.M. & Allsopp, R.C. The telomere hypothesis of cellular aging. Exp. Gerontol. 27, 375–382 (1992).

    Article  CAS  Google Scholar 

  6. Harley, C.B. & Villeponteau, B. Telomeres and telomerase in aging and cancer. Curr. Opin. Genet. Dev. 5, 249–255 (1995).

    Article  CAS  Google Scholar 

  7. Harley, C.B. Telomerase and cancer therapeutics. Nat. Rev. Cancer 8, 167–179 (2008).

    Article  CAS  Google Scholar 

  8. Bosoy, D., Peng, Y., Mian, I.S. & Lue, N.F. Conserved N-terminal motifs of telomerase reverse transcriptase required for ribonucleoprotein assembly in vivo. J. Biol. Chem. 278, 3882–3890 (2003).

    Article  CAS  Google Scholar 

  9. Bryan, T.M., Goodrich, K.J. & Cech, T.R. Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase. Mol. Cell 6, 493–499 (2000).

    Article  CAS  Google Scholar 

  10. Lai, C.K., Mitchell, J.R. & Collins, K. RNA binding domain of telomerase reverse transcriptase. Mol. Cell. Biol. 21, 990–1000 (2001).

    Article  CAS  Google Scholar 

  11. Drosopoulos, W.C. & Prasad, V.R. Telomerase-specific T motif is a restrictive determinant of repetitive reverse transcription by human telomerase. Mol. Cell. Biol. 30, 447–459 (2010).

    Article  CAS  Google Scholar 

  12. Rouda, S. & Skordalakes, E. Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding. Structure 15, 1403–1412 (2007).

    Article  CAS  Google Scholar 

  13. Hammond, P.W., Lively, T.N. & Cech, T.R. The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers. Mol. Cell. Biol. 17, 296–308 (1997).

    Article  CAS  Google Scholar 

  14. Jacobs, S.A., Podell, E.R. & Cech, T.R. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase. Nat. Struct. Mol. Biol. 13, 218–225 (2006).

    Article  CAS  Google Scholar 

  15. Wyatt, H.D., Lobb, D.A. & Beattie, T.L. Characterization of physical and functional anchor site interactions in human telomerase. Mol. Cell. Biol. 27, 3226–3240 (2007).

    Article  CAS  Google Scholar 

  16. Moriarty, T.J., Marie-Egyptienne, D.T. & Autexier, C. Functional organization of repeat addition processivity and DNA synthesis determinants in the human telomerase multimer. Mol. Cell. Biol. 24, 3720–3733 (2004).

    Article  CAS  Google Scholar 

  17. Wyatt, H.D., Tsang, A.R., Lobb, D.A. & Beattie, T.L. Human telomerase reverse transcriptase (hTERT) Q169 is essential for telomerase function in vitro and in vivo. PLoS One 4, e7176 (2009).

    Article  Google Scholar 

  18. Chen, J.L. & Greider, C.W. An emerging consensus for telomerase RNA structure. Proc. Natl. Acad. Sci. USA 101, 14683–14684 (2004).

    Article  CAS  Google Scholar 

  19. Lin, J. et al. A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein. Proc. Natl. Acad. Sci. USA 101, 14713–14718 (2004).

    Article  CAS  Google Scholar 

  20. Gilley, D., Lee, M.S. & Blackburn, E.H. Altering specific telomerase RNA template residues affects active site function. Genes Dev. 9, 2214–2226 (1995).

    Article  CAS  Google Scholar 

  21. 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).

    Article  CAS  Google Scholar 

  22. Lee, M.S. & Blackburn, E.H. Sequence-specific DNA primer effects on telomerase polymerization activity. Mol. Cell. Biol. 13, 6586–6599 (1993).

    Article  CAS  Google Scholar 

  23. Lingner, J., Hendrick, L.L. & Cech, T.R. Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev. 8, 1984–1998 (1994).

    Article  CAS  Google Scholar 

  24. Shippen-Lentz, D. & Blackburn, E.H. Functional evidence for an RNA template in telomerase. Science 247, 546–552 (1990).

    Article  CAS  Google Scholar 

  25. Autexier, C. & Greider, C.W. Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. Genes Dev. 9, 2227–2239 (1995).

    Article  CAS  Google Scholar 

  26. Harrington, L.A. & Greider, C.W. Telomerase primer specificity and chromosome healing. Nature 353, 451–454 (1991).

    Article  CAS  Google Scholar 

  27. Greider, C.W. Telomerase is processive. Mol. Cell. Biol. 11, 4572–4580 (1991).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Zaug, A.J., Podell, E.R. & Cech, T.R. Mutation in TERT separates processivity from anchor-site function. Nat. Struct. Mol. Biol. 15, 870–872 (2008).

    Article  CAS  Google Scholar 

  30. Finger, S.N. & Bryan, T.M. Multiple DNA-binding sites in Tetrahymena telomerase. Nucleic Acids Res. 36, 1260–1272 (2008).

    Article  CAS  Google Scholar 

  31. Osanai, M., Kojima, K.K., Futahashi, R., Yaguchi, S. & Fujiwara, H. Identification and characterization of the telomerase reverse transcriptase of Bombyx mori (silkworm) and Tribolium castaneum (flour beetle). Gene 376, 281–289 (2006).

    Article  CAS  Google Scholar 

  32. Richards, S. et al. The genome of the model beetle and pest Tribolium castaneum. Nature 452, 949–955 (2008).

    Article  CAS  Google Scholar 

  33. Bosoy, D. & Lue, N.F. Functional analysis of conserved residues in the putative “finger” domain of telomerase reverse transcriptase. J. Biol. Chem. 276, 46305–46312 (2001).

    Article  CAS  Google Scholar 

  34. Hossain, S., Singh, S. & Lue, N.F. Functional analysis of the C-terminal extension of telomerase reverse transcriptase. A putative “thumb” domain. J. Biol. Chem. 277, 36174–36180 (2002).

    Article  CAS  Google Scholar 

  35. Huang, H., Chopra, R., Verdine, G.L. & Harrison, S.C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669–1675 (1998).

    Article  CAS  Google Scholar 

  36. Sarafianos, S.G. et al. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA. EMBO J. 20, 1449–1461 (2001).

    Article  CAS  Google Scholar 

  37. Lue, N.F., Lin, Y.C. & Mian, I.S. A conserved telomerase motif within the catalytic domain of telomerase reverse transcriptase is specifically required for repeat addition processivity. Mol. Cell. Biol. 23, 8440–8449 (2003).

    Article  CAS  Google Scholar 

  38. Jacobo-Molina, A. et al. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 Å resolution shows bent DNA. Proc. Natl. Acad. Sci. USA 90, 6320–6324 (1993).

    Article  CAS  Google Scholar 

  39. Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A. & Steitz, T.A. Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 256, 1783–1790 (1992).

    Article  CAS  Google Scholar 

  40. Drosopoulos, W.C. & Prasad, V.R. The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity. Nucleic Acids Res. 35, 1155–1168 (2007).

    Article  CAS  Google Scholar 

  41. Rodgers, D.W. et al. The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 92, 1222–1226 (1995).

    Article  CAS  Google Scholar 

  42. Steitz, T.A. DNA and RNA polymerases: structural diversity and common mechanisms. Harvey Lect. 93, 75–93 (1997).

    PubMed  Google Scholar 

  43. Beese, L.S., Derbyshire, V. & Steitz, T.A. Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260, 352–355 (1993).

    Article  CAS  Google Scholar 

  44. Tantillo, C. et al. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. J. Mol. Biol. 243, 369–387 (1994).

    Article  CAS  Google Scholar 

  45. Cases-Gonzalez, C.E., Gutierrez-Rivas, M. & Menendez-Arias, L. Coupling ribose selection to fidelity of DNA synthesis. The role of Tyr-115 of human immunodeficiency virus type 1 reverse transcriptase. J. Biol. Chem. 275, 19759–19767 (2000).

    Article  CAS  Google Scholar 

  46. Ding, J. et al. Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 Å resolution. J. Mol. Biol. 284, 1095–1111 (1998).

    Article  CAS  Google Scholar 

  47. Steitz, T.A. DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–17398 (1999).

    Article  CAS  Google Scholar 

  48. Bressanelli, S., Tomei, L., Rey, F.A. & De Francesco, R. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J. Virol. 76, 3482–3492 (2002).

    Article  CAS  Google Scholar 

  49. Ling, H., Boudsocq, F., Woodgate, R. & Yang, W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102 (2001).

    Article  CAS  Google Scholar 

  50. Jeruzalmi, D., O'Donnell, M. & Kuriyan, J. Clamp loaders and sliding clamps. Curr. Opin. Struct. Biol. 12, 217–224 (2002).

    Article  CAS  Google Scholar 

  51. Chen, J.L. & Greider, C.W. Template boundary definition in mammalian telomerase. Genes Dev. 17, 2747–2752 (2003).

    Article  CAS  Google Scholar 

  52. Lai, C.K., Miller, M.C. & Collins, K. Template boundary definition in Tetrahymena telomerase. Genes Dev. 16, 415–420 (2002).

    Article  CAS  Google Scholar 

  53. Tzfati, Y., Fulton, T.B., Roy, J. & Blackburn, E.H. Template boundary in a yeast telomerase specified by RNA structure. Science 288, 863–867 (2000).

    Article  CAS  Google Scholar 

  54. Sasaki, T. & Fujiwara, H. Detection and distribution patterns of telomerase activity in insects. Eur. J. Biochem. 267, 3025–3031 (2000).

    Article  CAS  Google Scholar 

  55. Holton, J. & Alber, T. Automated protein crystal structure determination using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542 (2004).

    Article  CAS  Google Scholar 

  56. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131–1137 (2003).

    Article  Google Scholar 

  57. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  58. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  59. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank S.J. Gamblin and S. Pennell for helpful discussions. Funding for this project was generously provided by the Ellison Medical and the V Foundations as well as the Pennsylvania Department of Health.

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Authors and Affiliations

  1. Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, USA

    Meghan Mitchell, Andrew Gillis & Emmanuel Skordalakes

  2. Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan

    Mizuko Futahashi & Haruhiko Fujiwara

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  1. Meghan Mitchell
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  2. Andrew Gillis
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Contributions

E.S. designed the experiment plan, analyzed the data and wrote the manuscript; A.G. prepared the recombinant telomerase and carried out the reverse transcriptase assays; M.M. carried out the TRAP assays; H.F. and M.F. provided advice with the T. castaneum TRAP assays.

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Correspondence to Emmanuel Skordalakes.

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Mitchell, M., Gillis, A., Futahashi, M. et al. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat Struct Mol Biol 17, 513–518 (2010). https://doi.org/10.1038/nsmb.1777

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