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Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex

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SMC Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2004))

Abstract

The Mre11-Rad50-Nbs1 (MRN) complex coordinates the repair of DNA double-strand breaks, replication fork restart, meiosis, class-switch recombination, and telomere maintenance. As such, MRN is an essential molecular machine that has homologs in all organisms of life, from bacteriophage to humans. In human cells, MRN is a >500 kDa multifunctional complex that encodes DNA binding, ATPase, and both endonuclease and exonuclease activities. MRN also forms larger assemblies and interacts with multiple DNA repair and replication factors. The enzymatic properties of MRN have been the subject of intense research for over 20 years, and more recently, single-molecule biophysics studies are beginning to probe its many biochemical activities. Here, we describe the methods used to overexpress, fluorescently label, and visualize MRN and its activities on single molecules of DNA.

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References

  1. Vilenchik MM, Knudson AG (2003) Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer. Proc Natl Acad Sci U S A 100:12871–12876

    Article  CAS  Google Scholar 

  2. Schipler A, Iliakis G (2013) DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice. Nucleic Acids Res 41:7589–7605

    Article  CAS  Google Scholar 

  3. Mehta A, Haber JE (2014) Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb Perspect Biol 6:a016428

    Article  Google Scholar 

  4. Lam I, Keeney S (2015) Mechanism and regulation of meiotic recombination initiation. Cold Spring Harb Perspect Biol 7:a016634

    Article  Google Scholar 

  5. Jolly CJ, Cook AJL, Manis JP (2008) Fixing DNA breaks during class switch recombination. J Exp Med 205:509–513

    Article  CAS  Google Scholar 

  6. Doksani Y, de Lange T (2014) The role of double-strand break repair pathways at functional and dysfunctional telomeres. Cold Spring Harb Perspect Biol 6, a016576

    Article  Google Scholar 

  7. Aparicio T, Baer R, Gautier J (2014) DNA double-strand break repair pathway choice and cancer. DNA Repair 19:169–175

    Article  CAS  Google Scholar 

  8. Brown JS, O’Carrigan B, Jackson SP et al (2017) Targeting DNA repair in cancer: beyond PARP inhibitors. Cancer Discov 7:20–37

    Article  CAS  Google Scholar 

  9. Powell C, Mikropoulos C, Kaye SB et al (2010) Pre-clinical and clinical evaluation of PARP inhibitors as tumour-specific radiosensitisers. Cancer Treat Rev 36:566–575

    Article  CAS  Google Scholar 

  10. Lamarche BJ, Orazio NI, Weitzman MD (2010) The MRN complex in double-strand break repair and telomere maintenance. FEBS Lett 584:3682–3695

    Article  CAS  Google Scholar 

  11. Paull TT (2010) Making the best of the loose ends: Mre11/Rad50 complexes and Sae2 promote DNA double-strand break resection. DNA Repair (Amst) 9:1283–1291

    Article  CAS  Google Scholar 

  12. Cejka P (2015) DNA end resection: nucleases team up with the Right Partners to Initiate Homologous Recombination. J Biol Chem 290:22931–22938

    Article  CAS  Google Scholar 

  13. Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271

    Article  CAS  Google Scholar 

  14. Syed A, Tainer JA (2018) The MRE11-RAD50-NBS1 complex conducts the orchestration of damage signaling and outcomes to stress in DNA Replication and Repair. Annu Rev Biochem 87:263–294

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Deshpande RA, Lee J-H, Arora S et al (2016) Nbs1 converts the human Mre11/Rad50 nuclease complex into an endo/exonuclease machine specific for protein-DNA adducts. Mol Cell 64:593–606

    Article  CAS  Google Scholar 

  17. Paull TT, Deshpande RA (2014) The Mre11/Rad50/Nbs1 complex: recent insights into catalytic activities and ATP-driven conformational changes. Exp Cell Res 329:139–147

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Park YB, Hohl M, Padjasek M et al (2017) Eukaryotic Rad50 functions as a rod-shaped dimer. Nat Struct Mol Biol 24:248–257

    Article  CAS  Google Scholar 

  20. Hohl M, Kochańczyk T, Tous C et al (2015) Interdependence of the rad50 hook and globular domain functions. Mol Cell 57:479–491

    Article  CAS  Google Scholar 

  21. Hopfner KP, Karcher A, Shin DS et al (2000) Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 101:789–800

    Article  CAS  Google Scholar 

  22. Lim HS, Kim JS, Park YB et al (2011) Crystal structure of the Mre11-Rad50-ATPγS complex: understanding the interplay between Mre11 and Rad50. Genes Dev 25:1091–1104

    Article  CAS  Google Scholar 

  23. Paull TT, Gellert M (1999) Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. Genes Dev 13:1276–1288

    Article  CAS  Google Scholar 

  24. Difilippantonio S, Celeste A, Fernandez-Capetillo O et al (2005) Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models. Nat Cell Biol 7:675–685

    Article  CAS  Google Scholar 

  25. Tauchi H, Kobayashi J, Morishima K et al (2002) Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420:93–98

    Article  CAS  Google Scholar 

  26. Desai-Mehta A, Cerosaletti KM, Concannon P (2001) Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization. Mol Cell Biol 21:2184–2191

    Article  CAS  Google Scholar 

  27. Deshpande RA, Lee J-H, Paull TT (2017) Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites. Nucleic Acids Res 45:5255–5268

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Williams RS, Moncalian G, Williams JS et al (2008) Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair. Cell 135:97–109

    Article  CAS  Google Scholar 

  30. Liao S, Tammaro M, Yan H (2016) The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair. Nucleic Acids Res 44(12):5689–5701

    Article  CAS  Google Scholar 

  31. Lammens K, Bemeleit DJ, Möckel C et al (2011) The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair. Cell 145:54–66

    Article  CAS  Google Scholar 

  32. Möckel C, Lammens K, Schele A et al (2012) ATP driven structural changes of the bacterial Mre11:Rad50 catalytic head complex. Nucleic Acids Res 40:914–927

    Article  Google Scholar 

  33. Majka J, Alford B, Ausio J et al (2012) ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease. J Biol Chem 287:2328–2341

    Article  CAS  Google Scholar 

  34. Deshpande RA, Williams GJ, Limbo O et al (2014) ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J 33:482–500

    Article  CAS  Google Scholar 

  35. Shibata A, Moiani D, Arvai AS et al (2014) DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. Mol Cell 53:7–18

    Article  CAS  Google Scholar 

  36. Hopfner KP, Karcher A, Craig L et al (2001) Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell 105:473–485

    Article  CAS  Google Scholar 

  37. Hopfner KP, Karcher A, Shin D et al (2000) Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine. J Bacteriol 182:6036–6041

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Soniat MM, Myler LR, Schaub JM et al (2017) Next-generation DNA curtains for single-molecule studies of homologous recombination. Methods Enzymol 592:259–281

    Article  CAS  Google Scholar 

  40. Paull TT, Gellert M (1998) The 3′ to 5′ exonuclease activity of Mre11 facilitates repair of DNA double-strand breaks. Mol Cell 1:969–979

    Article  CAS  Google Scholar 

  41. Lee J-H, Mand MR, Deshpande RA et al (2013) Ataxia telangiectasia-mutated (ATM) kinase activity is regulated by ATP-driven conformational changes in the Mre11/Rad50/Nbs1 (MRN) complex. J Biol Chem 288:12840–12851

    Article  CAS  Google Scholar 

  42. Myler LR, Gallardo IF, Soniat MM et al (2017) Single-molecule imaging reveals how Mre11-Rad50-Nbs1 initiates DNA break repair. Mol Cell 67:891–898. e4

    Article  CAS  Google Scholar 

  43. Lee JY, Greene EC (2011) Assembly of recombinant nucleosomes on nanofabricated DNA curtains for single-molecule imaging. Methods Mol Biol 778:243–258

    Article  CAS  Google Scholar 

  44. Anand R, Ranjha L, Cannavo E et al (2016) Phosphorylated CtIP functions as a Co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection. Mol Cell 64:940–950

    Article  CAS  Google Scholar 

  45. Bieniossek C, Imasaki T, Takagi Y et al (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37:49–57

    Article  CAS  Google Scholar 

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Acknowledgments

We are indebted to Dr. Mauro Modesti for reagents. This work was supported by CPRIT (to I.J.F.), the National Institutes of Health (GM120554 and CA092584 to I.J.F.) and the Welch Foundation (F-l808 to I.J.F.). M.M.S. is supported by a postdoctoral fellowship, PF-17-169-01-DMC, from the American Cancer Society. L.R.M. is supported by the National Cancer Institute (CA212452). T.T.P. is an investigator of the Howard Hughes Medical Institute. I.J.F. is a CPRIT Scholar in cancer research.

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Authors and Affiliations

  1. Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

    Logan R. Myler, Michael M. Soniat, Xiaoming Zhang, Rajashree A. Deshpande, Tanya T. Paull & Ilya J. Finkelstein

  2. Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, USA

    Michael M. Soniat & Ilya J. Finkelstein

  3. The Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX, USA

    Xiaoming Zhang, Rajashree A. Deshpande & Tanya T. Paull

Authors
  1. Logan R. Myler
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  2. Michael M. Soniat
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  3. Xiaoming Zhang
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  4. Rajashree A. Deshpande
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  5. Tanya T. Paull
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  6. Ilya J. Finkelstein
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Correspondence to Ilya J. Finkelstein .

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Editors and Affiliations

  1. National Centre for Biological Sciences, Tata Institute of Fundamental Research (TIFR), Bangalore, India

    Anjana Badrinarayanan

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Myler, L.R., Soniat, M.M., Zhang, X., Deshpande, R.A., Paull, T.T., Finkelstein, I.J. (2019). Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex. In: Badrinarayanan, A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_20

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  • DOI: https://doi.org/10.1007/978-1-4939-9520-2_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9519-6

  • Online ISBN: 978-1-4939-9520-2

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