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Rudimentary G-quadruplex–based telomere capping in Saccharomyces cerevisiae

Abstract

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA–stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3′ overhang inhibits 5′→3′ resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo.

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Figure 1: Overexpression of the G4 DNA binding protein Stm1 rescues growth defects caused by telomere uncapping and is independent of RAD52-dependent homologous recombination.
Figure 2: Expression of two additional, distinct G4 DNA binding proteins rescues the cdc13-1 temperature-sensitive phenotype.
Figure 3: Loss of the SGS1 activities associated with G4 DNA binding and unwinding rescues cdc13-1 temperature sensitivity and is independent of RAD52-dependent homologous recombination.
Figure 4: Telomere-proximal single-stranded (ss) DNA accumulation at NPT (37 °C) is attenuated by two G4 DNA-stabilizing mechanisms.
Figure 5: Diminished rescue of cdc13-1 growth at SPT by sgs1 deletion in cells with mutant telomerase RNA templates that decrease QFP at telomeres.
Figure 6: G4 DNA-binding proteins and G4 DNA-forming sequences cooperate to inhibit end-resection by Exo1 in vitro.
Figure 7

References

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

    CAS  Article  PubMed  Google Scholar 

  2. Linger, B.R. & Price, C.M. Conservation of telomere protein complexes: shuffling through evolution. Crit. Rev. Biochem. Mol. Biol. 44, 434–446 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Garvik, B., Carson, M. & Hartwell, L. Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol. Cell. Biol. 15, 6128–6138 (1995).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Grandin, N., Damon, C. & Charbonneau, M. Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J. 20, 1173–1183 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Nugent, C.I., Hughes, T.R., Lue, N.F. & Lundblad, V. Cdc13p: A single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274, 249–252 (1996).

    CAS  Article  PubMed  Google Scholar 

  6. Burge, S., Parkinson, G.N., Hazel, P., Todd, A.K. & Neidle, S. Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res. 34, 5402–5415 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Risitano, A. & Fox, K.R. Influence of loop size on the stability of intramolecular DNA quadruplexes. Nucleic Acids Res. 32, 2598–2606 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Risitano, A. & Fox, K.R. Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes. Biochemistry 42, 6507–6513 (2003).

    CAS  Article  PubMed  Google Scholar 

  9. Venczel, E.A. & Sen, D. Parallel and antiparallel G-DNA structures from a complex telomeric sequence. Biochemistry 32, 6220–6228 (1993).

    CAS  Article  PubMed  Google Scholar 

  10. Paeschke, K., Simonson, T., Postberg, J., Rhodes, D. & Lipps, H.J. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat. Struct. Mol. Biol. 12, 847–854 (2005).

    CAS  Article  PubMed  Google Scholar 

  11. Zhang, M.L. et al. Yeast telomerase subunit Est1p has guanine quadruplex-promoting activity that is required for telomere elongation. Nat. Struct. Mol. Biol. 17, 202–209 (2010).

    CAS  Article  PubMed  Google Scholar 

  12. Zubko, M.K., Guillard, S. & Lydall, D. Exo1 and Rad24 differentially regulate generation of ssDNA at telomeres of Saccharomyces cerevisiae cdc13–1 mutants. Genetics 168, 103–115 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Hayashi, N. & Murakami, S. STM1, a gene which encodes a guanine quadruplex binding protein, interacts with CDC13 in Saccharomyces cerevisiae. Mol. Genet. Genomics 267, 806–813 (2002).

    CAS  Article  PubMed  Google Scholar 

  14. Frantz, J.D. & Gilbert, W. A yeast gene product, G4p2, with a specific affinity for quadruplex nucleic acids. J. Biol. Chem. 270, 9413–9419 (1995).

    CAS  Article  PubMed  Google Scholar 

  15. Van Dyke, M.W., Nelson, L.D., Weilbaecher, R.G. & Mehta, D.V. Stm1p, a G4 quadruplex and purine motif triplex nucleic acid-binding protein, interacts with ribosomes and subtelomeric Y′ DNA in Saccharomyces cerevisiae. J. Biol. Chem. 279, 24323–24333 (2004).

    CAS  Article  PubMed  Google Scholar 

  16. Nelson, L.D., Musso, M. & Van Dyke, M.W. The yeast STM1 gene encodes a purine motif triple helical DNA-binding protein. J. Biol. Chem. 275, 5573–5581 (2000).

    CAS  Article  PubMed  Google Scholar 

  17. Zubko, M.K. & Lydall, D. Linear chromosome maintenance in the absence of essential telomere-capping proteins. Nat. Cell Biol. 8, 734–740 (2006).

    CAS  Article  PubMed  Google Scholar 

  18. Huber, M.D., Lee, D.C. & Maizels, N. G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition. Nucleic Acids Res. 30, 3954–3961 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Huber, M.D., Duquette, M.L., Shiels, J.C. & Maizels, N.A. Conserved G4 DNA binding domain in RecQ family helicases. J. Mol. Biol. 358, 1071–1080 (2006).

    CAS  Article  PubMed  Google Scholar 

  20. Vodenicharov, M.D. & Wellinger, R.J. DNA degradation at unprotected telomeres in yeast is regulated by the CDK1 (Cdc28/Clb) cell-cycle kinase. Mol. Cell 24, 127–137 (2006).

    CAS  Article  PubMed  Google Scholar 

  21. Fernando, H. et al. Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression. Nucleic Acids Res. 37, 6716–6722 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Piazza, A. et al. Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae. Nucleic Acids Res. 38, 4337–4348 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. De Cian, A., DeLemos, E., Mergny, J.-L., Teulade-Fichou, M.-P. & Monchaud, D. Highly efficient G-quadruplex recognition by bisquinolinium compounds. J. Am. Chem. Soc. 129, 1856–1857 (2007).

    CAS  Article  PubMed  Google Scholar 

  24. Ren, J. & Chaires, J.B. Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 38, 16067–16075 (1999).

    CAS  Article  PubMed  Google Scholar 

  25. Smith, J.S. & Johnson, F.B. Isolation of G-quadruplex DNA using NMM-sepharose affinity chromatography. Methods Mol. Biol. 608, 207–221 (2010).

    CAS  Article  PubMed  Google Scholar 

  26. Li, Y., Geyer, R. & Sen, D. Recognition of anionic porphyrins by DNA aptamers. Biochemistry 35, 6911–6922 (1996).

    CAS  Article  PubMed  Google Scholar 

  27. Hershman, S.G. et al. Genomic distribution and functional analyses of potential G-quadruplex-forming sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 36, 144–156 (2008).

    CAS  Article  PubMed  Google Scholar 

  28. Zhu, Z., Chung, W.-H., Shim, E.Y., Lee, S.E. & Ira, G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134, 981–994 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Watt, P.M., Hickson, I.D., Borts, R.H. & Louis, E.J. SGS1, a homologue of the Bloom's and Werner's Syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae. Genetics 144, 935–945 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mimitou, E.P. & Symington, L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008).

    CAS  Article  PubMed  Google Scholar 

  31. Frei, C. & Gasser, S. The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci. Genes Dev. 14, 81–96 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Cobb, J.A., Bjergbaek, L., Shimada, K., Frei, C. & Gasser, S.M. DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. EMBO J. 22, 4325–4336 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Bernstein, D.A. & Keck, J.L. Conferring substrate specificity to DNA helicases: role of the RecQ HRDC domain. Structure 13, 1173–1182 (2005).

    CAS  Article  PubMed  Google Scholar 

  34. Mullen, J.R., Kaliraman, V. & Brill, S.J. Bipartite structure of the SGS1 DNA helicase in Saccharomyces cerevisiae. Genetics 154, 1101–1114 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Liu, Z. et al. The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins. Structure 7, 1557–1566 (1999).

    CAS  Article  PubMed  Google Scholar 

  36. Lu, J. et al. Human homologues of yeast helicase. Nature 383, 678–679 (1996).

    CAS  Article  PubMed  Google Scholar 

  37. Maringele, L. & Lydall, D. EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants. Genes Dev. 16, 1919–1933 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Lin, J., Smith, D.L. & Blackburn, E.H. Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response. Mol. Biol. Cell 15, 1623–1634 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Lane, A.N., Chaires, J.B., Gray, R.D. & Trent, J.O. Stability and kinetics of G-quadruplex structures. Nucleic Acids Res. 36, 5482–5515 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Förstemann, K., Hoss, M. & Lingner, J. Telomerase-dependent repeat divergence at the 3′ ends of yeast telomeres. Nucleic Acids Res. 28, 2690–2694 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Saccà, B., Lacroix, L. & Mergny, J.-L. The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides. Nucleic Acids Res. 33, 1182–1192 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Mergny, J.-L., Li, J., Lacroix, L., Amrane, S. & Chaires, J.B. Thermal difference spectra: a specific signature for nucleic acid structures. Nucleic Acids Res. 33, e138 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Kypr, J., Kejnovska, I., Renciuk, D. & Vorlickova, M. Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 37, 1713–1725 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Sandell, L.L. & Zakian, V.A. Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75, 729–739 (1993).

    CAS  Article  PubMed  Google Scholar 

  45. Addinall, S.G. et al. A genomewide suppressor and enhancer analysis of cdc13–1 reveals varied cellular processes influencing telomere capping in Saccharomyces cerevisiae. Genetics 180, 2251–2266 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Downey, M. et al. A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator. Cell 124, 1155–1168 (2006).

    CAS  Article  PubMed  Google Scholar 

  47. Ribeyre, C. et al. The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo. PLoS Genet. 5, e1000475 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Vallur, A.C. & Maizels, N. Distinct activities of exonuclease 1 and flap endonuclease 1 at telomeric G4 DNA. PLoS ONE 5, e8908 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Salas, T.R. et al. Human replication protein A unfolds telomeric G-quadruplexes. Nucleic Acids Res. 34, 4857–4865 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Tsai, Y.-C., Qi, H. & Liu, L.F. Protection of DNA ends by telomeric 3′ G-tail sequences. J. Biol. Chem. 282, 18786–18792 (2007).

    CAS  Article  PubMed  Google Scholar 

  51. Michelson, R.J., Rosenstein, S. & Weinert, T. A telomeric repeat sequence adjacent to a DNA double-stranded break produces an anticheckpoint. Genes Dev. 19, 2546–2559 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Bonetti, D., Martina, M., Clerici, M., Lucchini, G. & Longhese, M.P. Multiple pathways regulate 3′ overhang generation at S. cerevisiae telomeres. Mol. Cell 35, 70–81 (2009).

    CAS  Article  PubMed  Google Scholar 

  53. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Pedroso, I.M., Hayward, W. & Fletcher, T.M. The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures. Nucleic Acids Res. 37, 1541–1554 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. Sfeir, A. et al. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell 138, 90–103 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  57. Gomez, D. et al. The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells. Cancer Res. 66, 6908–6912 (2006).

    CAS  Article  PubMed  Google Scholar 

  58. Phatak, P. et al. Telomere uncapping by the G-quadruplex ligand RHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism. Br. J. Cancer 96, 1223–1233 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. Oganesian, L., Graham, M.E., Robinson, P.J. & Bryan, T.M. Telomerase recognizes G-quadruplex and linear DNA as distinct substrates. Biochemistry 46, 11279–11290 (2007).

    CAS  Article  PubMed  Google Scholar 

  60. Kozak, M.L. et al. Inactivation of the Sas2 histone acetyltransferase delays senescence driven by telomere dysfunction. EMBO J. 29, 158–170 (2010).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank the members of the Johnson and Yatsunyk labs for helpful discussions and comments on the manuscript; N. Maizels, M. Fry, K. Runge, V. Zakian, J.-L. Mergny, S. Berger, R. Marmorstein and P. Adams for discussions; S. Murakami (Kanazawa University), E. Blackburn (University of California, San Francisco), M. Charbonneau (École Normale Supérieure), S. Brill (Rutgers University), S. Gasser (Friedrich Miescher Institute) and M. Van Dyke (MD Anderson Cancer Center) for providing strains and plasmids, and D. Durocher (University of Toronto) for the Rad53 antibody. This work was supported by US National Institutes of Health grants R01 AG021521 (F.B.J.), P01 AG031862 (F.B.J.), T32 GM008216-22 (J.S.S.) and T32 GM07229 (J.S.S.), a Camille and Henry Dreyfus Faculty Startup Award (L.A.Y.), Research Corporation Award no. 7843 (L.A.Y.), a Deutscher Akademischer Austausch Dienst Fellowship (R.K.) and by a Cancer Research UK program grant (S.B.).

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Contributions

J.S.S. and F.B.J. conceived of and carried out experiments, interpreted results and wrote the manuscript. L.A.Y. and J.M.N. conducted and designed CD, thermal difference spectroscopy and fluorescence resonance energy transfer experiments, along with F.B.J. and J.S.S., and provided comments on the manuscript. R.K. and S.B. provided the HF1 cDNA and protein, designed and conducted CD and ELISA experiments (Supplementary Fig. 3) and provided comments on the manuscript. D.C.S. provided purified proteins and comments on the manuscript. D.M. and M.-P. T.-F. synthesized the bisquinolinium G4 DNA ligands and provided comments on the manuscript. Q.C., M.S.G. and L.A. carried out experiments.

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Correspondence to F Brad Johnson.

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Smith, J., Chen, Q., Yatsunyk, L. et al. Rudimentary G-quadruplex–based telomere capping in Saccharomyces cerevisiae. Nat Struct Mol Biol 18, 478–485 (2011). https://doi.org/10.1038/nsmb.2033

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