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Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair

Nature Structural & Molecular Biology volume 22, pages 158–166 (2015)Cite this article

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Abstract

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11–Rad50–Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.

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Figure 1: Tetrameric Ctp1 is an intrinsically disordered protein.
Figure 2: X-ray crystal structure of the Ctp1 N-terminal tetramerization domain.
Figure 3: Ctp1 binds DNA.
Figure 4: Identifying Ctp1 DNA-interaction motifs.
Figure 5: Ctp1 bridges DNA.
Figure 6: Sensitivity of Ctp1 THDD and RHR mutations to DNA-damaging agents.
Figure 7: Effects of Ctp1 THDD and RHR mutations on S. pombe DSB repair.
Figure 8: Overall model for Ctp1 molecular architecture from X-ray structures, SAXS and biophysical analysis.

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References

  1. Williams, R.S., Williams, J.S. & Tainer, J.A. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem. Cell Biol. 85, 509–520 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Pommier, Y. Drugging topoisomerases: lessons and challenges. ACS Chem. Biol. 8, 82–95 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. You, Z. et al. CtIP links DNA double-strand break sensing to resection. Mol. Cell 36, 954–969 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Williams, R.S. et al. Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand break processing and repair. Cell 139, 87–99 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Limbo, O. et al. Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination. Mol. Cell 28, 134–146 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Huertas, P., Cortes-Ledesma, F., Sartori, A.A., Aguilera, A. & Jackson, S.P. CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature 455, 689–692 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sartori, A.A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Huertas, P. & Jackson, S.P. Human CtIP mediates cell cycle control of DNA end resection and double strand break repair. J. Biol. Chem. 284, 9558–9565 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Stracker, T.H. & Petrini, J.H. The MRE11 complex: starting from the ends. Nat. Rev. Mol. Cell Biol. 12, 90–103 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, P.L. et al. Inactivation of CtIP leads to early embryonic lethality mediated by G1 restraint and to tumorigenesis by haploid insufficiency. Mol. Cell. Biol. 25, 3535–3542 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Qvist, P. et al. CtIP mutations cause Seckel and Jawad syndromes. PLoS Genet. 7, e1002310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhou, Y., Caron, P., Legube, G. & Paull, T.T. Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res. 42, e19 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Westmoreland, J.W. & Resnick, M.A. Coincident resection at both ends of random, gamma-induced double-strand breaks requires MRX (MRN), Sae2 (Ctp1), and Mre11-nuclease. PLoS Genet. 9, e1003420 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Langerak, P., Mejia-Ramirez, E., Limbo, O. & Russell, P. Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks. PLoS Genet. 7, e1002271 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mimitou, E.P. & Symington, L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Neale, M.J., Pan, J. & Keeney, S. Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053–1057 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Farah, J.A., Cromie, G.A. & Smith, G.R. Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic recombination. Proc. Natl. Acad. Sci. USA 106, 9356–9361 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Hartsuiker, E., Neale, M.J. & Carr, A.M. Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA. Mol. Cell 33, 117–123 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lengsfeld, B.M., Rattray, A.J., Bhaskara, V., Ghirlando, R. & Paull, T.T. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex. Mol. Cell 28, 638–651 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lobachev, K.S., Gordenin, D.A. & Resnick, M.A. The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell 108, 183–193 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Lloyd, J. et al. A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage. Cell 139, 100–111 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dubin, M.J. et al. Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif. J. Biol. Chem. 279, 26932–26938 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Dosztányi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Oates, M.E. et al. D2P2: database of disordered protein predictions. Nucleic Acids Res. 41, D508–D516 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Babu, M.M., Kriwacki, R.W. & Pappu, R.V. Structural biology: versatility from protein disorder. Science 337, 1460–1461 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Tompa, P. Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527–533 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Schiller, C.B. et al. Structure of Mre11–Nbs1 complex yields insights into ataxia-telangiectasia–like disease mutations and DNA damage signaling. Nat. Struct. Mol. Biol. 19, 693–700 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. You, Z., Chahwan, C., Bailis, J., Hunter, T. & Russell, P. ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Mol. Cell. Biol. 25, 5363–5379 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Akamatsu, Y. et al. Molecular characterization of the role of the Schizosaccharomyces pombe nip1+/ctp1+ gene in DNA double-strand break repair in association with the Mre11-Rad50-Nbs1 complex. Mol. Cell. Biol. 28, 3639–3651 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Williams, R.S. et al. Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair. Cell 135, 97–109 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hopfner, K.P. et al. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 418, 562–566 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Deshpande, R.A. et al. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J. 33, 482–500 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Andres, S.N. et al. A human XRCC4-XLF complex bridges DNA. Nucleic Acids Res. 40, 1868–1878 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hsiang, Y.H., Lihou, M.G. & Liu, L.F. Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 49, 5077–5082 (1989).

    CAS  PubMed  Google Scholar 

  35. Pommier, Y. & Cherfils, J. Interfacial inhibition of macromolecular interactions: nature's paradigm for drug discovery. Trends Pharmacol. Sci. 26, 138–145 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Povirk, L.F. Processing of damaged DNA ends for double-strand break repair in mammalian cells. ISRN Mol. Biol. 2012, 345805 (2012).

    Article  CAS  PubMed Central  Google Scholar 

  37. Kim, H.S. et al. Functional interactions between Sae2 and the Mre11 complex. Genetics 178, 711–723 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang, H. et al. CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks. J. Biol. Chem. 287, 21471–21480 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cannavo, E. & Cejka, P. Sae2 promotes dsDNA endonuclease activity within Mre11–Rad50–Xrs2 to resect DNA breaks. Nature 514, 122–125 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Eid, W. et al. DNA end resection by CtIP and exonuclease 1 prevents genomic instability. EMBO Rep. 11, 962–968 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhang, Y. & Jasin, M. An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway. Nat. Struct. Mol. Biol. 18, 80–84 (2011).

    Article  CAS  PubMed  Google Scholar 

  42. Moreno-Herrero, F. et al. Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA. Nature 437, 440–443 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Lobachev, K., Vitriol, E., Stemple, J., Resnick, M.A. & Bloom, K. Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex. Curr. Biol. 14, 2107–2112 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Chen, L., Trujillo, K., Ramos, W., Sung, P. & Tomkinson, A.E. Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol. Cell 8, 1105–1115 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Stols, L. et al. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. 25, 8–15 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Sreerama, N. & Woody, R.W. A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal. Biochem. 209, 32–44 (1993).

    Article  CAS  PubMed  Google Scholar 

  47. Sreerama, N., Venyaminov, S.Y. & Woody, R.W. Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8, 370–380 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Whitmore, L. & Wallace, B.A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–W673 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  50. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Thépaut, M. et al. Crystal structure of the coiled-coil dimerization motif of geminin: structural and functional insights on DNA replication regulation. J. Mol. Biol. 342, 275–287 (2004).

    Article  CAS  PubMed  Google Scholar 

  52. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  54. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  55. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

    Article  CAS  PubMed  Google Scholar 

  58. Tournier, S., Gachet, Y. & Hyams, J.S. Identification and preliminary characterization of p31, a new PSTAIRE-related protein in fission yeast. Yeast 13, 727–734 (1997).

    Article  CAS  PubMed  Google Scholar 

  59. Williams, J.S. et al. γH2A binds Brc1 to maintain genome integrity during S-phase. EMBO J. 29, 1136–1148 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Birren, B. & Lai, E. Pulsed Field Gel Electrophoresis: a Practical Guide (Academic Press, San Diego, 1993).

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Acknowledgements

Our studies are supported by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS) grants 1Z01ES102765 (R.S.W.) and 1Z01ES021016 (M.A.R.). We thank L. Pedersen of the NIEHS Collaborative crystallography group and the Advanced Photon Source (APS) Southeast Regional Collaborative Access Team (SER-CAT) for beamline access. Use of the APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We thank M. Junop (University of Western Ontario) for nicked plasmid substrate, J. Williams of the NIEHS protein microcharacterization core for MS analysis, R. Dutcher (NIEHS) for help with MALS analysis and G. Mueller (NIEHS) and B. Wallace (NIEHS) for comments on the manuscript.

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  1. Department of Health and Human Services, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, US National Institutes of Health, Research Triangle Park, North Carolina, USA.,

    Sara N Andres, C Denise Appel, James W Westmoreland, Jessica S Williams, Yvonne Nguyen, Patrick D Robertson, Michael A Resnick & R Scott Williams

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Contributions

S.N.A., R.S.W., M.A.R., J.W. and J.S.W. designed the experiments. S.N.A. and P.D.R. performed crystallization experiments. S.N.A. and R.S.W. solved and refined the Ctp1 X-ray structure. S.N.A. and R.S.W. carried out SAXS experiments. S.N.A., Y.N. and C.D.A. performed biochemical experiments. S.N.A., J.W., C.D.A. and J.S.W. performed S. pombe experiments. R.S.W. and S.N.A. wrote the manuscript with input from all authors. R.S.W. managed the project.

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Correspondence to R Scott Williams.

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Integrated supplementary information

Supplementary Figure 1 Ctp1 small-angle X-ray scattering (SAXS).

SAXS profiles of full length Ctp1 (blue), MBP-Ctp11­–60 (green) and MBP-Ctp115–60 (black). Inset: Guinier plots, colored as for scattering profiles, indicating the absence of aggregated protein in SAXS samples.

Supplementary Figure 2 A hydrophobic core stabilizes the Ctp1 tetrameric helical dimer-of-dimers (THDD) domain.

(a) Electrostatic surface potential representation illustrating the Ctp1 tetramer hydrophobic core and hydrophilic exterior. (b) Helical wheel diagram of Ctp1 parallel dimeric coiled-coil region. The heptad repeat with additional salt bridges (dotted lines) between coils is shown. Amino acid positions are numbered for “a” and “d” positions of the heptad repeat.

Supplementary Figure 3 Experimental electron density of the Ctp1 THDD domain.

A model phased, σ-A weighted 2Fo-Fc electron density map (blue, contoured at 1.0σ) was calculated from the initial molecular replacement polyalanine model solution. The corresponding σ-A weighted Fo-Fc map (green, contoured at 2.0σ) shows positive difference density for unmodeled helical regions, and amino acid side-chains.

Supplementary Figure 4 Oligomerization states of Ctp1 truncations and mutants.

(a) SEC-MALS traces of differential refractive index and molar mass for Ctp161–294 (blue) and Ctp1 THDD mutation (R32A K41A) (red). (b) SEC-MALS traces of differential refractive index and molar mass for N-terminal MBP-tagged Ctp11–60 (green) (as in Fig. 1d) and MBP-tagged Ctp11–60 (H11A W12A Y16A) (brown).

Supplementary Figure 5 Ctp1 binds DNA.

(a) Quantification of Ctp1–DNA binding. Mean values shown. Error bars, s.d. (n=3). (b) Ctp1FL binding variable lengths of double-stranded DNA. Arrow indicates 40­–50bp. (c) Purified Ctp1 deletion and internal deletion protein constructs. (d) Ctp1FL and Ctp1 protein halves binding DNA. (e) Ctp1FL and Ctp1 internal deletion DNA binding assays. WT, wildtype. Ctp1 concentrations expressed as tetrameric (tet) or monomeric (mono) as labeled. Experiments were repeated three times for (a), (b), (d), (e), with representative gels shown.

Supplementary Figure 6 Purified Ctp1FL N- and C-terminal mutations.

(a) Ctp1 N-terminal THDD domain point mutations used in DNA binding studies. (b) Ctp1 C-terminal point mutations used in DNA binding studies. WT, wildtype.

Supplementary Figure 7 Titration of DNA binding by Ctp1 mutants of the conserved RHR motif.

Ctp1 concentrations expressed as tetrameric (tet). Experiment was repeated 3 times with representative gels shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–4 (PDF 3777 kb)

Supplementary Data Set 1

Uncropped gels and blots (PDF 2633 kb)

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Andres, S., Appel, C., Westmoreland, J. et al. Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair. Nat Struct Mol Biol 22, 158–166 (2015). https://doi.org/10.1038/nsmb.2945

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