Abstract
Repressor activator protein 1 (RAP1) is the most highly conserved telomere protein. It is involved in protecting chromosome ends in fission yeast and promoting gene silencing in Saccharomyces cerevisiae, whereas it represses homology-directed recombination at telomeres in mammals. To understand how RAP1 has such diverse functions at telomeres, we solved the crystal or solution structures of the RAP1 C-terminal (RCT) domains of RAP1 from multiple organisms in complex with their respective protein-binding partners. Our analysis establishes RAP1RCT as an evolutionarily conserved protein-protein interaction module. In mammalian and fission yeast cells, this module interacts with TRF2 and Taz1, respectively, targeting RAP1 to chromosome ends for telomere protection. In contrast, S. cerevisiae RAP1 uses its RCT domain to recruit Sir3 to telomeres to mediate gene silencing. Together, our results show that, depending on the organism, the evolutionarily conserved RAP1 RCT motif has diverse functional roles at telomeres.
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References
de Lange, T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 19, 2100–2110 (2005).
Palm, W. & de Lange, T. How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42, 301–334 (2008).
Linger, B.R. & Price, C.M. Conservation of telomere protein complexes: shuffling through evolution. Crit. Rev. Biochem. Mol. Biol. 44, 434–446 (2009).
Li, B., Oestreich, S. & de Lange, T. Identification of human Rap1: implications for telomere evolution. Cell 101, 471–483 (2000).
Celli, G.B. & de Lange, T. DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat. Cell Biol. 7, 712–718 (2005).
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).
Guo, X. et al. Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis. EMBO J. 26, 4709–4719 (2007).
Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & de Lange, T. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283, 1321–1325 (1999).
van Steensel, B., Smogorzewska, A. & de Lange, T. TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413 (1998).
Bae, N.S. & Baumann, P.A. RAP1/TRF2 complex inhibits nonhomologous end-joining at human telomeric DNA ends. Mol. Cell 26, 323–334 (2007).
Sarthy, J., Bae, N.S., Scrafford, J. & Baumann, P. Human RAP1 inhibits non-homologous end joining at telomeres. EMBO J. 28, 3390–3399 (2009).
Martinez, P. et al. Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites. Nat. Cell Biol. 12, 768–780 (2010).
Sfeir, A., Kabir, S., van Overbeek, M., Celli, G.B. & de Lange, T. Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal. Science 327, 1657–1661 (2010).
Huet, J. et al. A general upstream binding factor for genes of the yeast translational apparatus. EMBO J. 4, 3539–3547 (1985).
Shore, D. RAP1: a protean regulator in yeast. Trends Genet. 10, 408–412 (1994).
Konig, P., Giraldo, R., Chapman, L. & Rhodes, D. The crystal structure of the DNA-binding domain of yeast RAP1 in complex with telomeric DNA. Cell 85, 125–136 (1996).
Moretti, P. & Shore, D. Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol. Cell. Biol. 21, 8082–8094 (2001).
Moretti, P., Freeman, K., Coodly, L. & Shore, D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 8, 2257–2269 (1994).
Wotton, D. & Shore, D. A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev. 11, 748–760 (1997).
Feeser, E.A. & Wolberger, C. Structural and functional studies of the Rap1 C-terminus reveal novel separation-of-function mutants. J. Mol. Biol. 380, 520–531 (2008).
Chikashige, Y. & Hiraoka, Y. Telomere binding of the Rap1 protein is required for meiosis in fission yeast. Curr. Biol. 11, 1618–1623 (2001).
Kanoh, J. & Ishikawa, F. spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast. Curr. Biol. 11, 1624–1630 (2001).
Miller, K.M., Ferreira, M.G. & Cooper, J.P. Taz1, Rap1 and Rif1 act both interdependently and independently to maintain telomeres. EMBO J. 24, 3128–3135 (2005).
Chen, Y. et al. A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins. Science 319, 1092–1096 (2008).
Deng, Y., Guo, X., Ferguson, D.O. & Chang, S. Multiple roles for MRE11 at uncapped telomeres. Nature 460, 914–918 (2009).
Wang, R.C., Smogorzewska, A. & de Lange, T. Homologous recombination generates T-loop-sized deletions at human telomeres. Cell 119, 355–368 (2004).
Wang, Y., Ghosh, G. & Hendrickson, E.A. Ku86 represses lethal telomere deletion events in human somatic cells. Proc. Natl. Acad. Sci. USA 106, 12430–12435 (2009).
Bailey, S.M., Goodwin, E.H., Meyne, J. & Cornforth, M.N. CO-FISH reveals inversions associated with isochromosome formation. Mutagenesis 11, 139–144 (1996).
Miyoshi, T., Kanoh, J., Saito, M. & Ishikawa, F. Fission yeast Pot1-Tpp1 protects telomeres and regulates telomere length. Science 320, 1341–1344 (2008).
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).
Boeke, J.D., LaCroute, F. & Fink, G.R. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197, 345–346 (1984).
Sussel, L. & Shore, D. Separation of transcriptional activation and silencing functions of the RAP1-encoded repressor/activator protein 1: isolation of viable mutants affecting both silencing and telomere length. Proc. Natl. Acad. Sci. USA 88, 7749–7753 (1991).
Rai, R. et al. The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres. EMBO J. 29, 2598–2610 (2010).
Wu, L. et al. Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell 126, 49–62 (2006).
d'Adda di Fagagna, F. et al. Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells. Curr. Biol. 11, 1192–1196 (2001).
Kim, H. et al. TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs. Nat. Struct. Mol. Biol. 16, 372–379 (2009).
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).
Horvath, M.P., Schweiker, V.L., Bevilacqua, J.M., Ruggles, J.A. & Schultz, S.C. Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963–974 (1998).
Lei, M., Podell, E.R., Baumann, P. & Cech, T.R. DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA. Nature 426, 198–203 (2003).
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. Nat. Struct. Mol. Biol. 11, 1223–1229 (2004).
Wang, F. et al. The POT1–TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506–510 (2007).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
La Fortelle, E.d. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).
Lamzin, V.S., Perrakis, A. & Wilson, K.S. The ARP/WARP suite for automated construction and refinement of protein models. in Int. Tables for Crystallography. Vol. F: Crystallography of Biological Macromolecules (Rossmann, M.G. & Arnold, E., eds.) 720–722 (Kluwer, Dordrecht, The Netherlands, 2001).
Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).
Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaaed, M. Improved methods for building protein models in electron density maps and the location of errors in these methods. Acta Crystallogr. A 47, 110–119 (1991).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Rieping, W. et al. ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23, 381–382 (2007).
Duggan, B.M., Legge, G.B., Dyson, H.J. & Wright, P.E. SANE (Structure Assisted NOE Evaluation): an automated model-based approach for NOE assignment. J. Biomol. NMR 19, 321–329 (2001).
Güntert, P., Mumenthaler, C. & Wuthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997).
Case, D.A. et al. The Amber biomolecular simulation programs. J. Comput. Chem. 26, 1668–1688 (2005).
Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).
Else, T. et al. Genetic p53 deficiency partially rescues the adrenocortical dysplasia phenotype at the expense of increased tumorigenesis. Cancer Cell 15, 465–476 (2009).
Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).
Bähler, J. et al. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).
Hentges, P., Van Driessche, B., Tafforeau, L., Vandenhaute, J. & Carr, A.M. Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast 22, 1013–1019 (2005).
Chikashige, Y. et al. Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation. J. Cell Biol. 187, 413–427 (2009).
Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).
Thomas, B.J. & Rothstein, R. Elevated recombination rates in transcriptionally active DNA. Cell 56, 619–630 (1989).
Acknowledgements
We thank J. Karlseder (Salk Institute for Biological Studies), L. Rudolph (University of Ulm) and M. Tarsounas (Cancer Research UK and University of Oxford) for providing valuable reagents. We thank F. Ishikawa (Kyoto University) for strains and Y. Ogiyama and K. Ishii (Osaka University) for technical support on PFGE. M.L. acknowledges financial support from the US National Institutes of Health (RO1 GM083015), the American Cancer Society and the Sidney Kimmel Foundation. M.L. is a Howard Hughes Medical Institute Early Career Scientist. S.C. acknowledges financial support from the US National Institute on Aging (RO1 AG028888), the US National Cancer Institute (RO1 CA129037), the Welch Foundation, the Susan G. Komen Race for the Cure Foundation, the Abraham and Phyllis Katz Foundation and the Michael Kadoorie Cancer Genetic Research Program. J.K. was supported by grants from Osaka University Life Science Young Independent Researcher Support Program (Japan Science and Technology Agency), the Japanese Ministry of Education, Culture, Sports, Science and Technology, Inamori Foundation, Astellas Foundation for Research on Metabolic Disorders and Takeda Science Foundation. Work in the laboratory of D.S. was supported by a grant from the Swiss National Fund (grant 31003A116716), by the Swiss National Fund Frontiers in Genetics NCCR program and by the Canton of Geneva. Use of Life Sciences Collaborative Access Team Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). Use of the Advanced Photon Source (APS) was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract no. DE-AC02-06CH11357).
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M.L., S.C., H.H., J.K. and D.S. designed the experiments. Y.C., R.R., Z.Z., C.R., Y.Y., H.Z., P.D., F.W. and H.T. conducted the experiments. M.L., S.C., H.H., J.K., D.S. and Y.H. analyzed the data. M.L. wrote the paper.
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Chen, Y., Rai, R., Zhou, ZR. et al. A conserved motif within RAP1 has diversified roles in telomere protection and regulation in different organisms. Nat Struct Mol Biol 18, 213–221 (2011). https://doi.org/10.1038/nsmb.1974
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DOI: https://doi.org/10.1038/nsmb.1974
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