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A topological mechanism for TRF2-enhanced strand invasion


Telomeres can fold into t-loops that may result from the invasion of the 3′ overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.

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Figure 1: TRF2 enhances telomeric strand invasion.
Figure 2: RPA and POT1 binding do not impair strand invasion.
Figure 3: TRF2 modifies DNA topology.
Figure 4: TRF2 stimulates invasion outside the telomeric tract.
Figure 5: TRF2 condenses telomeric DNA.
Figure 6: A topological model for telomeric strand invasion.

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  1. Chan, S.R. & Blackburn, E.H. Telomeres and telomerase. Phil. Trans. R. Soc. Lond. B 359, 109–121 (2004).

    Article  CAS  Google Scholar 

  2. Smogorzewska, A. & De Lange, T. Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208 (2004).

    Article  CAS  Google Scholar 

  3. Wenz, C. et al. Human telomerase contains two cooperating telomerase RNA molecules. EMBO J. 20, 3526–3534 (2001).

    Article  CAS  Google Scholar 

  4. Wang, Y. & Patel, D.J. Guanine residues in d(T2AG3) and d(T2G4) form parallel-stranded potassium cation stabilized G-quadruplexes with anti glycosidic torsion angles in solution. Biochemistry 31, 8112–8119 (1992).

    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. Stansel, R.M., de Lange, T. & Griffith, J.D. T-loop assembly in vitro involves binding of TRF2 near the 3′ telomeric overhang. EMBO J. 20, 5532–5540 (2001).

    Article  CAS  Google Scholar 

  7. Ancelin, K. et al. Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2. Mol. Cell. Biol. 22, 3474–3487 (2002).

    Article  CAS  Google Scholar 

  8. Wang, R.C., Smogorzewska, A. & de Lange, T. Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355–368 (2004).

    Article  CAS  Google Scholar 

  9. Dunham, M.A., Neumann, A.A., Fasching, C.L. & Reddel, R.R. Telomere maintenance by recombination in human cells. Nat. Genet. 26, 447–450 (2000).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Holloman, W.K., Wiegand, R., Hoessli, C. & Radding, C.M. Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination. Proc. Natl. Acad. Sci. USA 72, 2394–2398 (1975).

    Article  CAS  Google Scholar 

  15. Voloshin, O.N. et al. An eclectic DNA structure adopted by human telomeric sequence under superhelical stress and low pH. J. Biomol. Struct. Dyn. 9, 643–652 (1992).

    Article  CAS  Google Scholar 

  16. Henderson, E. et al. Structure, synthesis, and regulation of telomeres. Cancer Cells 6, 453–461 (1988).

    CAS  Google Scholar 

  17. Okabe, J., Eguchi, A., Masago, A., Hayakawa, T. & Nakanishi, M. TRF1 is a critical trans-acting factor required for de novo telomere formation in human cells. Hum. Mol. Genet. 9, 2639–2650 (2000).

    Article  CAS  Google Scholar 

  18. Cox, M.M. The bacterial RecA protein as a motor protein. Annu. Rev. Microbiol. 57, 551–577 (2003).

    Article  CAS  Google Scholar 

  19. Verdun, R.E. & Karlseder, J. The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres. Cell 127, 709–720 (2006).

    Article  CAS  Google Scholar 

  20. Court, R., Chapman, L., Fairall, L. & Rhodes, D. How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures. EMBO Rep. 6, 39–45 (2005).

    Article  CAS  Google Scholar 

  21. Fouche, N. et al. The basic domain of TRF2 directs binding to DNA junctions irrespective of the presence of TTAGGG repeats. J. Biol. Chem. 281, 37486–37495 (2006).

    Article  CAS  Google Scholar 

  22. Dantzer, F. et al. Functional interaction between poly(ADP-Ribose) polymerase 2 (PARP-2) and TRF2: PARP activity negatively regulates TRF2. Mol. Cell. Biol. 24, 1595–1607 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Opresko, P.L. et al. Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases. J. Biol. Chem. 277, 41110–41119 (2002).

    Article  CAS  Google Scholar 

  25. Shure, M., Pulleyblank, D.E. & Vinograd, J. The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity. Nucleic Acids Res. 4, 1183–1205 (1977).

    Article  CAS  Google Scholar 

  26. Waldmann, T., Eckerich, C., Baack, M. & Gruss, C. The ubiquitous chromatin protein DEK alters the structure of DNA by introducing positive supercoils. J. Biol. Chem. 277, 24988–24994 (2002).

    Article  CAS  Google Scholar 

  27. Musgrave, D.R., Sandman, K.M. & Reeve, J.N. DNA binding by the archaeal histone HMf results in positive supercoiling. Proc. Natl. Acad. Sci. USA 88, 10397–10401 (1991).

    Article  CAS  Google Scholar 

  28. Klungsoyr, H.K. & Skarstad, K. Positive supercoiling is generated in the presence of Escherichia coli SeqA protein. Mol. Microbiol. 54, 123–131 (2004).

    Article  CAS  Google Scholar 

  29. Rau, D.C., Gellert, M., Thoma, F. & Maxwell, A. Structure of the DNA gyrase-DNA complex as revealed by transient electric dichroism. J. Mol. Biol. 193, 555–569 (1987).

    Article  CAS  Google Scholar 

  30. Yoshimura, S.H., Maruyama, H., Ishikawa, F., Ohki, R. & Takeyasu, K. Molecular mechanisms of DNA end-loop formation by TRF2. Genes Cells 9, 205–218 (2004).

    Article  CAS  Google Scholar 

  31. Rivetti, C., Guthold, M. & Bustamante, C. Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J. 18, 4464–4475 (1999).

    Article  CAS  Google Scholar 

  32. Shin, M. et al. DNA looping-mediated repression by histone-like protein H-NS: specific requirement of Eσ70 as a cofactor for looping. Genes Dev. 19, 2388–2398 (2005).

    Article  CAS  Google Scholar 

  33. Belotserkovskii, B.P., Krasilnikova, M.M., Veselkov, A.G. & Frank-Kamenetskii, M.D. Kinetic trapping of H-DNA by oligonucleotide binding. Nucleic Acids Res. 20, 1903–1908 (1992).

    Article  CAS  Google Scholar 

  34. Budarf, M. & Blackburn, E. S1 nuclease sensitivity of a double-stranded telomeric DNA sequence. Nucleic Acids Res. 15, 6273–6292 (1987).

    Article  CAS  Google Scholar 

  35. Gilson, E., Muller, T., Sogo, J., Laroche, T. & Gasser, S.M. RAP1 stimulates single- to double-strand association of yeast telomeric DNA: implications for telomere-telomere interactions. Nucleic Acids Res. 22, 5310–5320 (1994).

    Article  CAS  Google Scholar 

  36. Huertas, D., Lipps, H. & Azorin, F. Characterization of the structural conformation adopted by (TTAGGG)n telomeric DNA repeats of different length in closed circular DNA. J. Biomol. Struct. Dyn. 12, 79–90 (1994).

    Article  CAS  Google Scholar 

  37. Lyamichev, V.I. et al. An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH. Nature 339, 634–637 (1989).

    Article  CAS  Google Scholar 

  38. Brunori, M. et al. TRF2 inhibition promotes anchorage-independent growth of telomerase-positive human fibroblasts. Oncogene 25, 990–997 (2006).

    Article  CAS  Google Scholar 

  39. 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 

  40. Zhu, X.D., Kuster, B., Mann, M., Petrini, J.H. & de Lange, T. Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat. Genet. 25, 347–352 (2000).

    Article  CAS  Google Scholar 

  41. Griffith, J., Bianchi, A. & de Lange, T. TRF1 promotes parallel pairing of telomeric tracts in vitro. J. Mol. Biol. 278, 79–88 (1998).

    Article  CAS  Google Scholar 

  42. Miller, K.M. & Cooper, J.P. The telomere protein Taz1 is required to prevent and repair genomic DNA breaks. Mol. Cell 11, 303–313 (2003).

    Article  CAS  Google Scholar 

  43. Kelleher, C., Kurth, I. & Lingner, J. Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro. Mol. Cell. Biol. 25, 808–818 (2005).

    Article  CAS  Google Scholar 

  44. Henricksen, L.A., Umbricht, C.B. & Wold, M.S. Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 (1994).

    CAS  PubMed  Google Scholar 

  45. Angelov, D., Novakov, E., Khochbin, S. & Dimitrov, S. Ultraviolet laser footprinting of histone H1(0)-four-way junction DNA complexes. Biochemistry 38, 11333–11339 (1999).

    Article  CAS  Google Scholar 

  46. Spassky, A. & Angelov, D. Influence of the local helical conformation on the guanine modifications generated from one-electron DNA oxidation. Biochemistry 36, 6571–6576 (1997).

    Article  CAS  Google Scholar 

  47. Lyubchenko, Y.L., Oden, P.I., Lampner, D., Lindsay, S.M. & Dunker, K.A. Atomic force microscopy of DNA and bacteriophage in air, water and propanol: the role of adhesion forces. Nucleic Acids Res. 21, 1117–1123 (1993).

    Article  CAS  Google Scholar 

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We thank R. Rahmouni for advice. This work was supported by grants from the Ligue Nationale contre le Cancer (“équipe labellisée”) and from “Région Centre”. C.L. is supported by fellowships from the Association pour la Recherche sur le Cancer.

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Correspondence to Eric Gilson.

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Supplementary information

Supplementary Fig. 1

Characterization of the telomeric strand invasion reaction. (PDF 144 kb)

Supplementary Fig. 2

DNA-binding properties of the TRF2 mutant forms used in this study. (PDF 223 kb)

Supplementary Fig. 3

Characterization of the TRF2-mediated strand invasion reaction. (PDF 109 kb)

Supplementary Fig. 4

TRF2 does not behave as a RecA-type protein. (PDF 101 kb)

Supplementary Fig. 5

TRF2, TRF2ΔB and TRF2ΔM form DNA complexes that do not migrate on polyacrylamide gels. (PDF 68 kb)

Supplementary Fig. 6

Topoisomers obtained in the topological assay do not originate from contaminations in the protein preparation. (PDF 65 kb)

Supplementary Fig. 7

TRF1, TRF2DM, TRF2DB and TRF2 do not create the same amount of supercoiling. (PDF 37 kb)

Supplementary Methods (PDF 103 kb)

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Amiard, S., Doudeau, M., Pinte, S. et al. A topological mechanism for TRF2-enhanced strand invasion. Nat Struct Mol Biol 14, 147–154 (2007).

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