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A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex

Nature Chemical Biology volume 4, pages 119–125 (2008)Cite this article

A Corrigendum to this article was published on 01 March 2009

This article has been updated

Abstract

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated Z-5-(4-hydroxybenzylidene)-2-imino-1,3-thiazolidin-4-one (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.

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Figure 1: Identification of mirin as an inhibitor of ATM activation by DSBs in X. laevis extracts.
Figure 2: Mirin inhibits MRN-dependent activation of ATM.
Figure 3: Mirin inhibits the nuclease activity of Mre11.
Figure 4: Mirin abolishes the G2/M checkpoint.
Figure 5: Mirin inhibits homology-dependent DNA repair in human cells.

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Change history

  • 13 February 2009

    In the version of this article initially published, the reported chemical structure and systematic name for mirin (compound 1), identified from a high-throughput screen, were incorrectly assigned in the commercial library. Investigations by Gartner, Pletnev and Eastman (Correspondence: Nat. Chem. Biol. 5, 129-130, 2009), which have been confirmed by us (Response: Nat. Chem. Biol. 5, 130, 2009), have identified the correct structure of mirin. The systematic name and chemical structure for mirin have been corrected in the HTML and PDF versions of the article, and in the graphical abstract and the PubChem database.

References

  1. Zhou, B.B. & Elledge, S.J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000).

    Article  CAS  Google Scholar 

  2. Kastan, M.B. & Bartek, J. Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004).

    Article  CAS  Google Scholar 

  3. Lavin, M.F. et al. ATM signaling and genomic stability in response to DNA damage. Mutat. Res. 569, 123–132 (2005).

    Article  CAS  Google Scholar 

  4. Bakkenist, C.J. & Kastan, M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    CAS  Google Scholar 

  5. 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  Google Scholar 

  6. Lee, J.H. & Paull, T.T. Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science 304, 93–96 (2004).

    Article  CAS  Google Scholar 

  7. Lee, J.H. & Paull, T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308, 551–554 (2005).

    Article  CAS  Google Scholar 

  8. Carson, C.T. et al. The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J. 22, 6610–6620 (2003).

    Article  CAS  Google Scholar 

  9. Difilippantonio, S. et al. Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models. Nat. Cell Biol. 7, 675–685 (2005).

    Article  CAS  Google Scholar 

  10. Dupre, A., Boyer-Chatenet, L. & Gautier, J. Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex. Nat. Struct. Mol. Biol. 13, 451–457 (2006).

    Article  CAS  Google Scholar 

  11. Kozlov, S.V. et al. Involvement of novel autophosphorylation sites in ATM activation. EMBO J. 25, 3504–3514 (2006).

    Article  CAS  Google Scholar 

  12. Gatei, M. et al. ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nat. Genet. 25, 115–119 (2000).

    Article  CAS  Google Scholar 

  13. Paull, T.T. & Lee, J.H. The Mre11/Rad50/Nbs1 complex and its role as a DNA double-strand break sensor for ATM. Cell Cycle 4, 737–740 (2005).

    Article  CAS  Google Scholar 

  14. Falck, J., Coates, J. & Jackson, S.P. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 434, 605–611 (2005).

    Article  CAS  Google Scholar 

  15. Uziel, T. et al. Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 22, 5612–5621 (2003).

    Article  CAS  Google Scholar 

  16. Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168 (2003).

    Article  CAS  Google Scholar 

  17. D'Amours, D. & Jackson, S.P. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling. Nat. Rev. Mol. Cell Biol. 3, 317–327 (2002).

    Article  CAS  Google Scholar 

  18. Paull, T.T. & Gellert, M. The 3′ to 5′ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. Mol. Cell 1, 969–979 (1998).

    Article  CAS  Google Scholar 

  19. Haber, J.E. The many interfaces of Mre11. Cell 95, 583–586 (1998).

    Article  CAS  Google Scholar 

  20. Zhou, B.B. & Bartek, J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat. Rev. Cancer 4, 216–225 (2004).

    Article  CAS  Google Scholar 

  21. Costanzo, V. et al. Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage. Mol. Cell 6, 649–659 (2000).

    Article  CAS  Google Scholar 

  22. Costanzo, V. et al. Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. Mol. Cell 8, 137–147 (2001).

    Article  CAS  Google Scholar 

  23. Peterson, J.R., Lokey, R.S., Mitchison, T.J. & Kirschner, M.W. A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions. Proc. Natl. Acad. Sci. USA 98, 10624–10629 (2001).

    Article  CAS  Google Scholar 

  24. Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117–120 (2004).

    Article  CAS  Google Scholar 

  25. Wignall, S.M. et al. Identification of a novel protein regulating microtubule stability through a chemical approach. Chem. Biol. 11, 135–146 (2004).

    Article  CAS  Google Scholar 

  26. Costanzo, V., Paull, T., Gottesman, M. & Gautier, J. Mre11 assembles linear DNA fragments into DNA damage signaling complexes. PLoS Biol. 2, E110 (2004).

    Article  Google Scholar 

  27. Komarov, P.G. et al. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285, 1733–1737 (1999).

    Article  CAS  Google Scholar 

  28. Mayer, T.U. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974 (1999); comment 286, 913–914 (1999).

    Article  CAS  Google Scholar 

  29. Cimprich, K.A., Shin, T.B., Keith, C.T. & Schreiber, S.L. cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc. Natl. Acad. Sci. USA 93, 2850–2855 (1996).

    Article  CAS  Google Scholar 

  30. Kitagawa, R., Bakkenist, C.J., McKinnon, P.J. & Kastan, M.B. Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway. Genes Dev. 18, 1423–1438 (2004).

    Article  CAS  Google Scholar 

  31. Lee, J.H. & Paull, T.T. Purification and biochemical characterization of ataxia-telangiectasia mutated and Mre11/Rad50/Nbs1. Methods Enzymol. 408, 529–539 (2006).

    Article  CAS  Google Scholar 

  32. Bhaskara, V. et al. Rad50 adenylate kinase activity regulates DNA tethering activities of Mre11/Rad50 complexes. Mol. Cell 25, 647–661 (2007).

    Article  CAS  Google Scholar 

  33. Trenz, K., Smith, E., Smith, S. & Costanzo, V. ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks. EMBO J. 25, 1764–1774 (2006).

    Article  CAS  Google Scholar 

  34. Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159 (2004).

    Article  CAS  Google Scholar 

  35. Pierce, A.J., Johnson, R.D., Thompson, L.H. & Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13, 2633–2638 (1999).

    Article  CAS  Google Scholar 

  36. Esashi, F. et al. CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature 434, 598–604 (2005).

    Article  CAS  Google Scholar 

  37. Arthur, L.M. et al. Structural and functional analysis of Mre11–3. Nucleic Acids Res. 32, 1886–1893 (2004).

    Article  CAS  Google Scholar 

  38. Symington, L.S. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66, 630–670 (2002).

    Article  CAS  Google Scholar 

  39. Smythe, C. & Newport, J.W. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol. 35, 449–468 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank H. Lindsay for Chk2 antibody and B. Stockwell, O. Haccard and the members of the Gautier laboratory for helpful discussions. We thank G. Smith (KuDOS Pharmaceuticals) for the inhibitor KU-55933. We are indebted to J. Decatur and Y. Itagaki of the Chemistry Department for providing NMR and mass spectral analysis of mirin. This work was supported by US National Institutes of Health grants CA95866 and CA92245 (J. Gautier), CA97403 (R. Baer) and GM54668 (M. Jasin).

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Author notes

  1. Aude Dupré & Louise Boyer-Chatenet

    Present address: Present addresses: Centre National de la Recherche Scientifique-Unité Mixte de recherche 7622, case 24, 4 place Jussieu 75005 Paris, France (A.D.) and Service de cardiologie, 184, rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France (L.B.-C.).,

  2. Aude Dupré and Louise Boyer-Chatenet: These authors contributed equally to this work.

Authors and Affiliations

  1. Columbia University, Institute for Cancer Genetics, Irving Cancer Research Center, 1130 St. Nicholas Avenue, Room 602, New York, 10032, New York, USA

    Aude Dupré, Louise Boyer-Chatenet, Rose M Sattler, Ami P Modi, Richard Baer & Jean Gautier

  2. Department of Molecular Genetics and Microbiology and the Institute of Cellular and Molecular Biology, University of Texas, 1 University Station, A4800, Austin, 78712, Texas, USA

    Ji-Hoon Lee, Matthew L Nicolette & Tanya T Paull

  3. Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., MSC 7315, Bethesda, 20892, Maryland, USA

    Levy Kopelovich

  4. Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 430 East 67th Street, New York, 10065, New York, USA

    Maria Jasin

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Correspondence to Jean Gautier.

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Dupré, A., Boyer-Chatenet, L., Sattler, R. et al. A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex. Nat Chem Biol 4, 119–125 (2008). https://doi.org/10.1038/nchembio.63

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