Abstract

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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Acknowledgements

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).

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Author notes

  1. These authors contributed equally: Maria Victoria Botuyan, Gaofeng Cui, Pascal Drané.

Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

    • Maria Victoria Botuyan
    • , Gaofeng Cui
    • , James R. Thompson
    • , Benoît Bragantini
    • , Debiao Zhao
    •  & Georges Mer
  2. Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Pascal Drané
    • , Alexandre Detappe
    • , Marie Eve Brault
    • , Nishita Parnandi
    • , Shweta Chaubey
    •  & Dipanjan Chowdhury
  3. Genome Integrity Laboratory, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK

    • Catarina Oliveira
    •  & J. Ross Chapman
  4. Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    • Dipanjan Chowdhury
  5. Broad Institute of Harvard and MIT, Cambridge, MA, USA

    • Dipanjan Chowdhury

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Contributions

M.V.B., G.C., P.D., D.C., and G.M. conceived the study. M.V.B., G.C., P.D., C.O., A.D., S.C., M.E.B., N.P., J.R.T., B.B., D.Z., J.R.C., D.C., and G.M. performed the experiments and/or analyzed the data. G.M. wrote the manuscript with extensive input from M.V.B., G.C., P.D., and D.C. All authors edited the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Dipanjan Chowdhury or Georges Mer.

Integrated supplementary information

  1. Supplementary Figure 1 X-ray structures of TIRR–53BP1 and NUDT16TI–53BP1.

    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

  2. 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.

  3. 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

  4. Supplementary Figure 4 Class switch recombination in stimulated B cells.

    Shown are representative flow cytometry plots of the data presented in Fig. 5h

  5. Supplementary Figure 5 Comparison of the X-ray structures of TIRR–53BP1 and NUDT16TI–53BP1.

    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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https://doi.org/10.1038/s41594-018-0083-z