In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Mattarocci, S., Hafner, L., Lezaja, A., Shyian, M. & Shore, D. Rif1: a conserved regulator of DNA replication and repair hijacked by telomeres in yeasts. Front. Genet. 7, 45 (2016).
Mattarocci, S. et al. Rif1 controls DNA replication timing in yeast through the PP1 phosphatase Glc7. Cell Rep. 7, 62–69 (2014).
Davé, A., Cooley, C., Garg, M. & Bianchi, A. Protein phosphatase 1 recruitment by Rif1 regulates DNA replication origin firing by counteracting DDK activity. Cell Rep. 7, 53–61 (2014).
Hiraga, S. et al. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex. Genes Dev. 28, 372–383 (2014).
Cornacchia, D. et al. Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells. EMBO J. 31, 3678–3690 (2012).
Hayano, M. et al. Rif1 is a global regulator of timing of replication origin firing in fission yeast. Genes Dev. 26, 137–150 (2012).
Yamazaki, S., Hayano, M. & Masai, H. Replication timing regulation of eukaryotic replicons: Rif1 as a global regulator of replication timing. Trends Genet. 29, 449–460 (2013).
Yamazaki, S. et al. Rif1 regulates the replication timing domains on the human genome. EMBO J. 31, 3667–3677 (2012).
Peace, J.M., Ter-Zakarian, A. & Aparicio, O.M. Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome. PLoS One 9, e98501 (2014).
König, 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).
Hardy, C.F., Sussel, L. & Shore, D. A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation. Genes Dev. 6, 801–814 (1992).
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).
Shi, T. et al. Rif1 and Rif2 shape telomere function and architecture through multivalent Rap1 interactions. Cell 153, 1340–1353 (2013).
Bianchi, A. & Shore, D. Increased association of telomerase with short telomeres in yeast. Genes Dev. 21, 1726–1730 (2007).
Ribeyre, C. & Shore, D. Anticheckpoint pathways at telomeres in yeast. Nat. Struct. Mol. Biol. 19, 307–313 (2012).
Sabourin, M., Tuzon, C.T. & Zakian, V.A. Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae. Mol. Cell 27, 550–561 (2007).
Hirano, Y., Fukunaga, K. & Sugimoto, K. Rif1 and rif2 inhibit localization of tel1 to DNA ends. Mol. Cell 33, 312–322 (2009).
Silverman, J., Takai, H., Buonomo, S.B., Eisenhaber, F. & de Lange, T. Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint. Genes Dev. 18, 2108–2119 (2004).
Xu, L. & Blackburn, E.H. Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules. J. Cell Biol. 167, 819–830 (2004).
Buonomo, S.B.C., Wu, Y., Ferguson, D. & de Lange, T. Mammalian Rif1 contributes to replication stress survival and homology-directed repair. J. Cell Biol. 187, 385–398 (2009).
Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).
Wilson, M.D. et al. The structural basis of modified nucleosome recognition by 53BP1. Nature 536, 100–103 (2016).
Escribano-Díaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883 (2013).
Chapman, J.R. et al. RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection. Mol. Cell 49, 858–871 (2013).
Di Virgilio, M. et al. Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching. Science 339, 711–715 (2013).
Zimmermann, M., Lottersberger, F., Buonomo, S.B., Sfeir, A. & de Lange, T. 53BP1 regulates DSB repair using Rif1 to control 5′ end resection. Science 339, 700–704 (2013).
Martina, M., Bonetti, D., Villa, M., Lucchini, G. & Longhese, M.P. Saccharomyces cerevisiae Rif1 cooperates with MRX-Sae2 in promoting DNA-end resection. EMBO Rep. 15, 695–704 (2014).
Ira, G. & Nussenzweig, A. A new Riff: Rif1 eats its cake and has it too. EMBO Rep. 15, 622–624 (2014).
Sreesankar, E., Senthilkumar, R., Bharathi, V., Mishra, R.K. & Mishra, K. Functional diversification of yeast telomere associated protein, Rif1, in higher eukaryotes. BMC Genomics 13, 255 (2012).
Finn, R.D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44 D1, D279–D285 (2016).
Anbalagan, S., Bonetti, D., Lucchini, G. & Longhese, M.P. Rif1 supports the function of the CST complex in yeast telomere capping. PLoS Genet. 7, e1002024 (2011).
Xue, Y., Rushton, M.D. & Maringele, L. A novel checkpoint and RPA inhibitory pathway regulated by Rif1. PLoS Genet. 7, e1002417 (2011).
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).
Haruki, H., Nishikawa, J. & Laemmli, U.K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31, 925–932 (2008).
Lee, S.E., Pâques, F., Sylvan, J. & Haber, J.E. Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths. Curr. Biol. 9, 767–770 (1999).
Moore, J.K. & Haber, J.E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2164–2173 (1996).
Horigome, C. et al. PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL. Genes Dev. 30, 931–945 (2016).
Boulton, S.J. & Jackson, S.P. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res. 24, 4639–4648 (1996).
Mages, G.J., Feldmann, H.M. & Winnacker, E.L. Involvement of the Saccharomyces cerevisiae HDF1 gene in DNA double-strand break repair and recombination. J. Biol. Chem. 271, 7910–7915 (1996).
Siede, W., Friedl, A.A., Dianova, I., Eckardt-Schupp, F. & Friedberg, E.C. The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination. Genetics 142, 91–102 (1996).
Clikeman, J.A., Khalsa, G.J., Barton, S.L. & Nickoloff, J.A. Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms. Genetics 157, 579–589 (2001).
Zhang, Y. et al. Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination. Nat. Struct. Mol. Biol. 14, 639–646 (2007).
Walker, J.R., Corpina, R.A. & Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614 (2001).
Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Zierhut, C. & Diffley, J.F. Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J. 27, 1875–1885 (2008).
Cronin, C.N., Lim, K.B. & Rogers, J. Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells. Protein Sci. 16, 2023–2029 (2007).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).
Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).
Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. in Macromolecular Crystallography Protocols Vol. 2 (ed. Doublié, S.) 215–230 (Humana Press, 2007).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Cowtan, K. dm': an automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Protein Crystallography 31, 34–38 (1994).
Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).
Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).
Hohn, M. et al. SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007).
Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Rose, M.D., Winston, F. & Hieter, P. Methods in Yeast Genetics: A Laboratory Course Manual (Cold Spring Harbor Press, 1990).
Longtine, M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).
Stuckey, S., Mukherjee, K. & Storici, F. In vivo site-specific mutagenesis and gene collage using the delitto perfetto system in yeast Saccharomyces cerevisiae. Methods Mol. Biol. 745, 173–191 (2011).
Horigome, C. et al. SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice. Mol. Cell 55, 626–639 (2014).
We thank the following Technology Platform members of the Friedrich Miescher Institute: A. Graff-Meyer and A. Schenk (Electron Microscopy Facility) for assistance in the collection of the negative-stain EM data and analysis, D. Hess (Proteomics and Protein Analysis) for support with protein analyses, H. Gut and J. Keusch (Protein Structure) for support with protein crystallization and crystallographic data collection. Crystallographic experiments were performed at beamline X06SA and X06DA of the Swiss Light Source, Paul Scherrer Institut, Switzerland. J.K.R. was supported by a Boehringer Ingelheim Fonds PhD fellowship, and L.H. by an Excellence Master fellowship from the University of Geneva. Work in the laboratory of U.R. is supported by the Swiss Cancer League & Swiss Cancer Research and the Novartis Research Foundation. The laboratory of N.H.T. is supported by the Novartis Research Foundation. This project received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 666068, N.H.T.). Work in the laboratory of D.S. and N.H.T. was supported by the Swiss National Science Foundation (grant 31003A_149463 to D.S., and Sinergia grant CRSII3_160734 to D.S. and N.H.T.). We thank S. Gasser and I. Hickson for fruitful discussions and S. Gasser (Friedrich Miescher Institute) for providing yeast strains. We would like to thank all members of the Gasser, Thomä, Rass, and Shore laboratories for valuable input and technical assistance.
The authors declare no competing financial interests.
About this article
Cite this article
Mattarocci, S., Reinert, J., Bunker, R. et al. Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends. Nat Struct Mol Biol 24, 588–595 (2017) doi:10.1038/nsmb.3420
Nucleic Acids Research (2019)
Current Genetics (2019)
Fungal Biology (2019)
Rif1 promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization activities
Scientific Reports (2019)
Nature Communications (2019)