Abstract
Telomeres are eukaryotic protein–DNA complexes found at the ends of linear chromosomes that are essential for maintaining genome integrity and are implicated in cellular aging and cancer. The guanine (G)-rich strand of telomeric DNA, usually elongated by the telomerase reverse transcriptase, can form a higher-order structure known as a G-quadruplex in vitro and in vivo. Several factors that promote or resolve G-quadruplexes have been identified, but the functional importance of these structures for telomere maintenance is not well understood. Here we show that the yeast telomerase subunit Est1p, known to be involved in telomerase recruitment to telomeres, can convert single-stranded telomeric G-rich DNA into a G-quadruplex structure in vitro in a Mg2+-dependent manner. Cells carrying Est1p mutants deficient in G-quadruplex formation in vitro showed gradual telomere shortening and cellular senescence, indicating a positive regulatory role for G-quadruplex in the maintenance of telomere length.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
McEachern, M.J., Krauskopf, A. & Blackburn, E.H. Telomeres and their control. Annu. Rev. Genet. 34, 331–358 (2000).
Smogorzewska, A. & de Lange, T. Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208 (2004).
Greider, C.W. & Blackburn, E.H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405–413 (1985).
Wellinger, R.J., Wolf, A.J. & Zakian, V.A. Saccharomyces telomeres acquire single-strand TG1–3 tails late in S phase. Cell 72, 51–60 (1993).
Lundblad, V. & Szostak, J.W. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57, 633–643 (1989).
Lingner, J. et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276, 561–567 (1997).
Singer, M.S. & Gottschling, D.E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266, 404–409 (1994).
Lingner, J., Cech, T.R., Hughes, T.R. & Lundblad, V. Three ever shorter telomere (EST) genes are dispensable for in vitro yeast telomerase activity. Proc. Natl. Acad. Sci. USA 94, 11190–11195 (1997).
Liao, X.H., Zhang, M.L., Yang, C.P., Xu, L.X. & Zhou, J.Q. Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast. Biochem. J. 390, 169–176 (2005).
Hughes, T.R., Evans, S.K., Weilbaecher, R.G. & Lundblad, V. The Est3 protein is a subunit of yeast telomerase. Curr. Biol. 10, 809–812 (2000).
Virta-Pearlman, V., Morris, D.K. & Lundblad, V. Est1 has the properties of a single-stranded telomere end–binding protein. Genes Dev. 10, 3094–3104 (1996).
Seto, A.G., Livengood, A.J., Tzfati, Y., Blackburn, E.H. & Cech, T.R. A bulged stem tethers Est1p to telomerase RNA in budding yeast. Genes Dev. 16, 2800–2812 (2002).
Pennock, E., Buckley, K. & Lundblad, V. Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104, 387–396 (2001).
Qi, H. & Zakian, V.A. The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein. Genes Dev. 14, 1777–1788 (2000).
Evans, S.K. & Lundblad, V. Est1 and Cdc13 as comediators of telomerase access. Science 286, 117–120 (1999).
Evans, S.K. & Lundblad, V. The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. Genetics 162, 1101–1115 (2002).
Taggart, A.K., Teng, S.C. & Zakian, V.A. Est1p as a cell cycle–regulated activator of telomere-bound telomerase. Science 297, 1023–1026 (2002).
Johnson, J.E., Smith, J.S., Kozak, M.L. & Johnson, F.B. In vivo veritas: using yeast to probe the biological functions of G-quadruplexes. Biochimie 90, 1250–1263 (2008).
Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. USA 98, 8572–8577 (2001).
Paeschke, K., Simonsson, 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).
Fang, G. & Cech, T.R. The β subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA. Cell 74, 875–885 (1993).
Venczel, E.A. & Sen, D. Parallel and antiparallel G-DNA structures from a complex telomeric sequence. Biochemistry 32, 6220–6228 (1993).
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).
Giraldo, R., Suzuki, M., Chapman, L. & Rhodes, D. Promotion of parallel DNA quadruplexes by a yeast telomere binding protein: a circular dichroism study. Proc. Natl. Acad. Sci. USA 91, 7658–7662 (1994).
Johnson, F.B. et al. The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase. EMBO J. 20, 905–913 (2001).
Sun, H., Bennett, R.J. & Maizels, N. The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs. Nucleic Acids Res. 27, 1978–1984 (1999).
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).
Oganesian, L., Moon, I.K., Bryan, T.M. & Jarstfer, M.B. Extension of G-quadruplex DNA by ciliate telomerase. EMBO J. 25, 1148–1159 (2006).
Zahler, A.M., Williamson, J.R., Cech, T.R. & Prescott, D.M. Inhibition of telomerase by G-quartet DNA structures. Nature 350, 718–720 (1991).
Sen, D.G.W. A sodium-potassium switch in the formation of four-stranded G4-DNA. Nature 344, 410–414 (1990).
Sen, D. & Gilbert, W. Guanine quartet structures. Methods Enzymol. 211, 191–199 (1992).
Kretsinger, R.H. & Nockolds, C.E. Carp muscle calcium-binding protein. II. Structure determination and general description. J. Biol. Chem. 248, 3313–3326 (1973).
Strynadka, N.C. & James, M.N. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58, 951–998 (1989).
Matsuura, I. et al. A site-directed mutagenesis study of yeast calmodulin. J. Biochem. 109, 190–197 (1991).
Lundblad, V. & Blackburn, E.H. An alternative pathway for yeast telomere maintenance rescues est1– senescence. Cell 73, 347–360 (1993).
Diede, S.J. & Gottschling, D.E. Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases α and δ. Cell 99, 723–733 (1999).
Fisher, T.S., Taggart, A.K. & Zakian, V.A. Cell cycle–dependent regulation of yeast telomerase by Ku. Nat. Struct. Mol. Biol. 11, 1198–1205 (2004).
Chan, A., Boule, J.B. & Zakian, V.A. Two pathways recruit telomerase to Saccharomyces cerevisiae telomeres. PLoS Genet. 4, e1000236 (2008).
Osterhage, J.L., Talley, J.M. & Friedman, K.L. Proteasome-dependent degradation of Est1p regulates the cell cycle–restricted assembly of telomerase in Saccharomyces cerevisiae. Nat. Struct. Mol. Biol. 13, 720–728 (2006).
Schramke, V. et al. RPA regulates telomerase action by providing Est1p access to chromosome ends. Nat. Genet. 36, 46–54 (2004).
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).
Singh, S.M. & Lue, N.F. Ever shorter telomere 1 (EST1)-dependent reverse transcription by Candida telomerase in vitro: evidence in support of an activating function. Proc. Natl. Acad. Sci. USA 100, 5718–5723 (2003).
Prescott, J. & Blackburn, E.H. Functionally interacting telomerase RNAs in the yeast telomerase complex. Genes Dev. 11, 2790–2800 (1997).
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).
Luke, B. et al. The Rat1p 5′ to 3′ exonuclease degrades telomeric repeat–containing RNA and promotes telomere elongation in Saccharomyces cerevisiae. Mol. Cell 32, 465–477 (2008).
Croy, J.E., Podell, E.R. & Wuttke, D.S. A new model for Schizosaccharomyces pombe telomere recognition: the telomeric single-stranded DNA-binding activity of Pot11–389. J. Mol. Biol. 361, 80–93 (2006).
Paeschke, K., Juranek, S., Simonsson, T., Hempel, A., Rhodes, D. & Lipps, H.J. Telomerase recruitment by the telomere end binding protein-β facilitates G-quadruplex DNA unfolding in ciliates. Nat. Struct. Mol. Biol. 15, 598–604 (2008).
Xin, H., Liu, D., Wan, M., Safari, A., Kim, H., Sun, W., O'Connor, M.S. & Songyang, Z. TPP1 is a homologue of ciliate TEBP-β and interacts with POT1 to recruit telomerase. Nature 445, 559–562 (2007).
Wang, F., Podell, E.R., Zaug, A.J., Yang, Y., Baciu, P., Cech, T.R. & Lei, M. The POT1–TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506–510 (2007).
Fang, G. & Cech, T.R. Oxytricha telomere-binding protein: DNA-dependent dimerization of the α and β subunits. Proc. Natl. Acad. Sci. USA 90, 6056–6060 (1993).
Froelich-Ammon, S.J., Dickinson, B.A., Bevilacqua, J.M., Schultz, S.C. & Cech, T.R. Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha. Genes Dev. 12, 1504–1514 (1998).
Lin, Y.C., Shih, J.W., Hsu, C.L. & Lin, J.J. Binding and partial denaturing of G-quartet DNA by Cdc13p of Saccharomyces cerevisiae. J. Biol. Chem. 276, 47671–47674 (2001).
Lei, M., Zaug, A.J., Podell, E.R. & Cech, T.R. Switching human telomerase on and off with hPOT1 protein in vitro. J. Biol. Chem. 280, 20449–20456 (2005).
Park, P.U., Defossez, P.A. & Guarente, L. Effects of mutations in DNA repair genes on formation of ribosomal DNA circles and life span in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 3848–3856 (1999).
Zhou, J.Q.T.C., So, A.G. & Downey, K.M. Purification and characterization of the catalytic subunit of human DNA polymerase δ expressed in baculovirus-infected insect cells. J. Biol. Chem. 271, 29740–29745 (1996).
Cogoi, S. & Xodo, L.E. G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription. Nucleic Acids Res. 34, 2536–2549 (2006).
Tsukamoto, Y., Mitsuoka, C., Terasawa, M., Ogawa, H. & Ogawa, T. Xrs2p regulates Mre11p translocation to the nucleus and plays a role in telomere elongation and meiotic recombination. Mol. Biol. Cell 16, 597–608 (2005).
Friedman, K.L. & Cech, T.R. Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants. Genes Dev. 13, 2863–2874 (1999).
Livengood, A.J., Zaug, A.J. & Cech, T.R. Essential regions of Saccharomyces cerevisiae telomerase RNA: separate elements for Est1p and Est2p interaction. Mol. Cell. Biol. 22, 2366–2374 (2002).
Gottschling, D.E., Aparicio, O.M., Billington, B.L. & Zakian, V.A. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751–762 (1990).
Acknowledgements
We are grateful to Y. Tsukamoto (Iwate College of Nursing), D. Gottschling (Fred Hutchinson Cancer Research Center), S.-C. Teng (Department of Microbiology, College of Medicine, National Taiwan University), V. Zakian (Department of Molecular Biology, Princeton University) and V. Lundblad (The Salk Institute for Biological Studies) for providing the plasmids and yeast strains. We thank L.-X. Xu for antibody preparations, B.A. Lenzmeier and J. Peng for their critical reading of our manuscript and Y. Hu for creating the image of Figure 7. This work is supported by a Chinese Academy of Sciences–Max Planck Society Professorship and by grants from the Natural Science Foundation of China (NSFC30630018) and the Ministry of Science and Technology (2005CB522402/2007CB914502) to J.-Q.Z.
Author information
Authors and Affiliations
Contributions
M.-L.Z. and X.-J.T. proposed the project, performed most of the experiments, interpreted the data, designed experiments and wrote the manuscript; X.-H.F, B.O.Z., J.W., X.-H.L and Q.-J.L performed some of the experiments; N.S. and J.D. helped with the structure modeling; J.-Q.Z. supervised the project, designed the experiments, interpreted the data and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Table 1 and Supplementary Figures 1–9 (PDF 2727 kb)
Rights and permissions
About this article
Cite this article
Zhang, ML., Tong, XJ., Fu, XH. 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). https://doi.org/10.1038/nsmb.1760
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1760
This article is cited by
-
Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage
BMC Biology (2021)
-
The regulation and functions of DNA and RNA G-quadruplexes
Nature Reviews Molecular Cell Biology (2020)
-
Modulation of yeast telomerase activity by Cdc13 and Est1 in vitro
Scientific Reports (2016)
-
Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length
Nature Communications (2015)
-
Telomeric G-quadruplexes are a substrate and site of localization for human telomerase
Nature Communications (2015)