Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.
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Botuyan, M. V. et al. Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).
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).
Mattiroli, F. et al. RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell 150, 1182–1195 (2012).
Gatti, M. et al. A novel ubiquitin mark at the N-terminal tail of histone H2As targeted by RNF168 ubiquitin ligase. Cell Cycle 11, 2538–2544 (2012).
Panier, S. & Boulton, S. J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).
Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).
Bouwman, P. et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat. Struct. Mol. Biol. 17, 688–695 (2010).
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).
Feng, L., Fong, K. W., Wang, J., Wang, W. & Chen, J. RIF1 counteracts BRCA1-mediated end resection during DNA repair. J. Biol. Chem. 288, 11135–11143 (2013).
Canny, M. D. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol. 36, 95–102 (2018).
Manis, J. P. et al. 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5, 481–487 (2004).
Ward, I. M. et al. 53BP1 is required for class switch recombination. J. Cell Biol. 165, 459–464 (2004).
Drané, P. et al. TIRR regulates 53BP1 by masking its histone methyl-lysine binding function. Nature 543, 211–216 (2017).
Zhang, A., Peng, B., Huang, P., Chen, J. & Gong, Z. The p53-binding protein 1-Tudor-interacting repair regulator complex participates in the DNA damage response. J. Biol. Chem. 292, 6461–6467 (2017).
Taylor, M. J. & Peculis, B. A. Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme. Nucleic Acids Res. 36, 6021–6034 (2008).
He, C. et al. High-resolution mapping of RNA-binding regions in the nuclear proteome of embryonic stem cells. Mol. Cell 64, 416–430 (2016).
Francia, S. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235 (2012).
Burger, K. et al. Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage. J. Cell Biol. 216, 2373–2389 (2017).
Michelini, F. et al. Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks. Nat. Cell Biol. 19, 1400–1411 (2017).
Hu, Q., Botuyan, M. V., Cui, G., Zhao, D. & Mer, G. Mechanisms of ubiquitin-nucleosome recognition and regulation of 53BP1 chromatin recruitment by RNF168/169 and RAD18. Mol. Cell 66, 473–487.e9 (2017).
Ward, I. et al. The tandem BRCT domain of 53BP1 is not required for its repair function. J. Biol. Chem. 281, 38472–38477 (2006).
Zgheib, O., Pataky, K., Brugger, J. & Halazonetis, T. D. An oligomerized 53BP1 Tudor domain suffices for recognition of DNA double-strand breaks. Mol. Cell Biol. 29, 1050–1058 (2009).
Tang, J. et al. Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nat. Struct. Mol. Biol. 20, 317–325 (2013).
Tong, Q. et al. Structural plasticity of methyllysine recognition by the tandem Tudor domain of 53BP1. Structure 23, 312–321 (2015).
Kim, H. et al. Crystal structure of syndesmos and its interaction with Syndecan-4 proteoglycan. Biochem. Biophys. Res. Commun. 463, 762–767 (2015).
Trésaugues, L. et al. Structural basis for the specificity of human NUDT16 and its regulation by inosine monophosphate. PLoS One 10, e0131507 (2015).
Lu, G. et al. hNUDT16: a universal decapping enzyme for small nucleolar RNA and cytoplasmic mRNA. Protein Cell 2, 64–73 (2011).
Iyama, T., Abolhassani, N., Tsuchimoto, D., Nonaka, M. & Nakabeppu, Y. NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest. Nucleic Acids Res. 38, 4834–4843 (2010).
Abolhassani, N. et al. NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals. Nucleic Acids Res. 38, 2891–2903 (2010).
Daniels, C. M., Thirawatananond, P., Ong, S. E., Gabelli, S. B. & Leung, A. K. Nudix hydrolases degrade protein-conjugated ADP-ribose. Sci. Rep. 5, 18271 (2015).
Palazzo, L. et al. Processing of protein ADP-ribosylation by Nudix hydrolases. Biochem. J. 468, 293–301 (2015).
Zong, D. et al. Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining. Nucleic Acids Res. 43, 4950–4961 (2015).
Dimitrova, N., Chen, Y. C., Spector, D. L. & de Lange, T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456, 524–528 (2008).
Margalef, P. et al. Stabilization of reversed replication forks by telomerase drives telomere catastrophe. Cell 172, 439–453.e14 (2018).
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).
Smogorzewska, A., Karlseder, J., Holtgreve-Grez, H., Jauch, A. & de Lange, T. DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2. Curr. Biol. 12, 1635–1644 (2002).
Bothmer, A. et al. Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1. Mol. Cell 42, 319–329 (2011).
Callen, E. et al. 53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions. Cell 153, 1266–1280 (2013).
Pryde, F. et al. 53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin. J. Cell Sci. 118, 2043–2055 (2005).
Lee, J., Thompson, J. R., Botuyan, M. V. & Mer, G. Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor. Nat. Struct. Mol. Biol. 15, 109–111 (2008).
Mallette, F. A. et al. RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites. EMBO J. 31, 1865–1878 (2012).
Acs, K. et al. The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks. Nat. Struct. Mol. Biol. 18, 1345–1350 (2011).
Poulsen, M., Lukas, C., Lukas, J., Bekker-Jensen, S. & Mailand, N. Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks. J. Cell Biol. 197, 189–199 (2012).
Panier, S. et al. Tandem protein interaction modules organize the ubiquitin-dependent response to DNA double-strand breaks. Mol. Cell 47, 383–395 (2012).
Chen, J., Feng, W., Jiang, J., Deng, Y. & Huen, M. S. Ring finger protein RNF169 antagonizes the ubiquitin-dependent signaling cascade at sites of DNA damage. J. Biol. Chem. 287, 27715–27722 (2012).
Jacquet, K. et al. The TIP60 complex regulates bivalent chromatin recognition by 53BP1 through direct H4K20me binding and H2AK15 acetylation. Mol. Cell 62, 409–421 (2016).
Yang, Q., Gilmartin, G. M. & Doublié, S. Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3′ processing. Proc. Natl Acad. Sci. USA 107, 10062–10067 (2010).
Orthwein, A. et al. Mitosis inhibits DNA double-strand break repair to guard against telomere fusions. Science 344, 189–193 (2014).
Lee, D. H. et al. Dephosphorylation enables the recruitment of 53BP1 to double-strand DNA breaks. Mol. Cell 54, 512–525 (2014).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).
Delano, W. F. The PyMOL Molecular Graphics System, version 1.3r1. (Schrodinger, LLC, New York, 2010).
Brown, P. H., Balbo, A. & Schuck, P. Using prior knowledge in the determination of macromolecular size-distributions by analytical ultracentrifugation. Biomacromolecules 8, 2011–2024 (2007).
Drané, P., Ouararhni, K., Depaux, A., Shuaib, M. & Hamiche, A. The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3. Genes Dev. 24, 1253–1265 (2010).
Karlseder, J., Smogorzewska, A. & de Lange, T. Senescence induced by altered telomere state, not telomere loss. Science 295, 2446–2449 (2002).
Ward, I. M., Minn, K., van Deursen, J. & Chen, J. p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice. Mol. Cell. Biol. 23, 2556–2563 (2003).
We are very grateful to R. Alkire, N. Duke, and J. Lazarz at Argonne National Laboratory for their outstanding assistance. X-ray diffraction data were collected at Argonne National Laboratory, Structural Biology Center (SBC) at the Advanced Photon Source. SBC is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. This research was supported by NIH grants R01 CA132878, R01 GM116829, and P50 CA136393 (Mayo Clinic Ovarian Cancer SPORE developmental project) to G.M.; and by NIH grants R01 CA208244 and R01CA142698, DoD grant W81XWH-15-0564/OC140632, a Leukemia and Lymphoma Society Scholar grant, and the Claudia Adams Barr Program in Innovative Basic Cancer Research to D.C. M.V.B. was supported by DoD grant W81XWH-16-1-0391 and a Liz Tilberis award from the Ovarian Cancer Research Fund Alliance. G.C. received a Fellowship Award from the Mayo Clinic Cancer Center Fraternal Order of Eagles Funds. J.R.C. and C.O. were supported by a Cancer Research UK Career Development Fellowship Grant (C52690/A19270).
The authors declare no competing interests.
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Integrated supplementary information
a, The four TIRR and two 53BP1-Tudor molecules in the asymmetric unit are shown. b, The two NUDT16TI and two 53BP1-Tudor molecules in the asymmetric unit are shown
Supplementary Figure 2 Conformations of TIRR 53BP1-binding loop and corresponding loop region in NUDT16.
Left: Amino acid sequence alignment of TIRR 53BP1-binding loop with corresponding region of NUDT16. Right: Structural overlay of the loop regions in TIRR (blue) and NUDT16 (gray). Highlighted are Pro104 and Arg105 in NUDT16 and key 53BP1-interacting residues Pro105 and Arg107 in TIRR. 53BP1 residues interacting with TIRR Pro105 and Arg107 are also shown.
Supplementary Figure 3 A flexible loop in TIRR (residues 101–107) is highly specific for 53BP1 interaction.
Silver-stained gel (a) and immunoblot (b) of TIRR-FH and TIRR-Loop-FH partner proteins purified from the soluble nuclear extract of U2OS cells for mass spectrometric analysis. TIRR-Loop-FH corresponds to TIRR-FH mutated to harbor the loop region of NUDT16 (see Supplementary Fig. 2). Mass spectrometry data are in Supplementary Table 1
Shown are representative flow cytometry plots of the data presented in Fig. 5h
a, Overlay of the TIRR–53BP1 and NUDT16TI–53BP1 structures with TIRR shown in blue, NUDT16TI in light blue, and 53BP1 in orange. The C-terminal α-helices in TIRR homodimer (shown in gray) do not exist in NUDT16TI. b, Details of the TIRR–53BP1 and NUDT16TI–53BP1 binding interfaces illustrating the remarkable similarity between the two complexes. Same color-coding as in a.
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Botuyan, M.V., Cui, G., Drané, P. et al. Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein. Nat Struct Mol Biol 25, 591–600 (2018). https://doi.org/10.1038/s41594-018-0083-z
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