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Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends

Abstract

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.

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Figure 1: Crystal structures of Rif1-NTD in isolation and in complex with DNA.
Figure 2: The Rif1-NTD HOOK domain binds DNA.
Figure 3: Rif1-NTD is an effector of telomere homeostasis at native and critically short telomeres.
Figure 4: Rif1-NTD attenuates end resection at chromosome-internal DSBs to promote DNA repair by NHEJ.
Figure 5: Rif1-NTD controls the fate of DNA ends at telomeres and chromosome breaks.

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Acknowledgements

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.

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U.R., N.H.T., and D.S. conceived this study. J.K.R and T.S. expressed and purified recombinant proteins with help from M.F. and produced crystals with help from R.D.B. J.K.R., T.S., and R.D.B. collected crystallographic data. R.D.B. carried out the crystallographic analysis and interpreted the results. J.K.R. designed, performed, and analyzed electromobility shift assays with help from D.K. J.K.R. designed and performed the negative-stain EM experiments and analyzed the results with help from S.C. S.M. designed, performed, and analyzed western blot and ChIP experiments with help from M.S. and L.H. S.M. designed, performed, and analyzed the assay to measure checkpoint activation, the assays to score the viability of ts mutants, and the Southern blots to assess telomere length. S.M. and G.A.F. performed and analyzed the qPCR experiments to measure ssDNA formed by DNA end resection. G.A.F. designed and analyzed the colony outgrowth assays to score NHEJ efficiency and DNA-damage resistance; data collection was performed with blinding by G.A.F. and D.K. G.A.F. designed, performed, and analyzed DSB stability experiments by Southern blotting with help from D.K. S.M., J.K.R., R.D.B., and G.A.F. contributed equally to this work. All the authors discussed the data. U.R., N.H.T., and D.S. wrote the manuscript with input from S.M., J.K.R., R.D.B., and G.A.F.

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Correspondence to David Shore or Nicolas H Thomä or Ulrich Rass.

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Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Note 1 (PDF 1975 kb)

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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). https://doi.org/10.1038/nsmb.3420

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