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DNA Repair pp 225-244 | Cite as

Assembling the Human Resectosome on DNA Curtains

  • Michael M. Soniat
  • Logan R. Myler
  • Ilya J. FinkelsteinEmail author
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1999)

Abstract

DNA double-strand breaks (DSBs) are a potentially lethal DNA lesions that disrupt both the physical and genetic continuity of the DNA duplex. Homologous recombination (HR) is a universally conserved genome maintenance pathway that initiates via nucleolytic processing of the broken DNA ends (resection). Eukaryotic DNA resection is catalyzed by the resectosome—a multicomponent molecular machine consisting of the nucleases DNA2 or Exonuclease 1 (EXO1), Bloom’s helicase (BLM), the MRE11-RAD50-NBS1 (MRN) complex, and additional regulatory factors. Here, we describe methods for purification and single-molecule imaging and analysis of EXO1, DNA2, and BLM. We also describe how to adapt resection assays to the high-throughput single-molecule DNA curtain assay. By organizing hundreds of individual molecules on the surface of a microfluidic flowcell, DNA curtains visualize protein complexes with the required spatial and temporal resolution to resolve the molecular choreography during critical DNA-processing reactions.

Key words

DNA curtains Homologous recombination Bloom’s syndrome helicase (BLM) DNA nuclease (DNA2) Exonuclease 1 (EXO1) 
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Notes

Acknowledgments

We are indebted to Drs. Mauro Modesti and Tanya Paull for plasmids, cell pellets, and other reagents. This work was supported by the National Institutes of Health (GM120554 and CA092584) 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).

References

  1. 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 100:12871–12876CrossRefGoogle Scholar
  2. 2.
    Vilenchik MM, Knudson AG (2006) Radiation dose-rate effects, endogenous DNA damage, and signaling resonance. Proc Natl Acad Sci U S A 103:17874–17879CrossRefGoogle Scholar
  3. 3.
    Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40:179–204CrossRefGoogle Scholar
  4. 4.
    Symington LS (2016) Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol 51:195–212CrossRefGoogle Scholar
  5. 5.
    Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271CrossRefGoogle Scholar
  6. 6.
    Jasin M, Rothstein R (2013) Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol 5:a012740CrossRefGoogle Scholar
  7. 7.
    Cannavo E, Cejka P (2014) Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks. Nature 514:122–125CrossRefGoogle Scholar
  8. 8.
    Paull TT, Gellert M (1998) The 3′ to 5′ exonuclease activity of Mre11 facilitates repair of DNA double-strand breaks. Mol Cell 1:969–979CrossRefGoogle Scholar
  9. 9.
    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 53:7–18CrossRefGoogle Scholar
  10. 10.
    Lukas C, Melander F, Stucki M et al (2004) Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J 23:2674–2683CrossRefGoogle Scholar
  11. 11.
    Stracker TH, Petrini JHJ (2011) The MRE11 complex: starting from the ends. Nat Rev Mol Cell Biol 12:90–103CrossRefGoogle Scholar
  12. 12.
    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 288:12840–12851CrossRefGoogle Scholar
  13. 13.
    Tauchi H, Kobayashi J, Morishima K et al (2002) Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420:93–98CrossRefGoogle Scholar
  14. 14.
    Desai-Mehta A, Cerosaletti KM, Concannon P (2001) Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization. Mol Cell Biol 21:2184–2191CrossRefGoogle Scholar
  15. 15.
    Williams RS, Dodson GE, Limbo O et al (2009) Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand break processing and repair. Cell 139:87–99CrossRefGoogle Scholar
  16. 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 64:593–606CrossRefGoogle Scholar
  17. 17.
    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 64:940–950CrossRefGoogle Scholar
  18. 18.
    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. e4CrossRefGoogle Scholar
  19. 19.
    Mimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455:770–774CrossRefGoogle Scholar
  20. 20.
    Zhu Z, Chung W-H, Shim EY et al (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134:981–994CrossRefGoogle Scholar
  21. 21.
    Garcia V, Phelps SE, Gray S et al (2011) Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1. Nature 479:241–244CrossRefGoogle Scholar
  22. 22.
    Cejka P, Cannavo E, Polaczek P et al (2010) DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467:112–116CrossRefGoogle Scholar
  23. 23.
    Nimonkar AV, Genschel J, Kinoshita E et al (2011) BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev 25:350–362CrossRefGoogle Scholar
  24. 24.
    Nimonkar AV, Ozsoy AZ, Genschel J et al (2008) Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair. Proc Natl Acad Sci U S A 105:16906–16911CrossRefGoogle Scholar
  25. 25.
    Niu H, Chung W-H, Zhu Z et al (2010) Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature 467:108–111CrossRefGoogle Scholar
  26. 26.
    Gravel S, Chapman JR, Magill C et al (2008) DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev 22:2767–2772CrossRefGoogle Scholar
  27. 27.
    Mimitou EP, Symington LS (2011) DNA end resection—Unraveling the tail. DNA Repair 10:344–348CrossRefGoogle Scholar
  28. 28.
    Tomimatsu N, Mukherjee B, Deland K et al (2012) Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions. DNA Repair 11:441–448CrossRefGoogle Scholar
  29. 29.
    Myler LR, Gallardo IF, Zhou Y et al (2016) Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc Natl Acad Sci U S A 113:e1170–e1179CrossRefGoogle Scholar
  30. 30.
    Gallardo IF, Pasupathy P, Brown M et al (2015) High-throughput universal DNA curtain arrays for single-molecule fluorescence imaging. Langmuir 31:10310–10317CrossRefGoogle Scholar
  31. 31.
    Levikova M, Pinto C, Cejka P (2017) The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection. Genes Dev 31:493–502CrossRefGoogle Scholar
  32. 32.
    Levikova M, Klaue D, Seidel R et al (2013) Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity. Proc Natl Acad Sci U S A 110:E1992–E2001CrossRefGoogle Scholar
  33. 33.
    Pinto C, Kasaciunaite K, Seidel R et al (2016) Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases. elife 5:e18574CrossRefGoogle Scholar
  34. 34.
    Szankasi P, Smith GR (1992) A DNA exonuclease induced during meiosis of Schizosaccharomyces pombe. J Biol Chem 267:3014–3023PubMedGoogle Scholar
  35. 35.
    Wu P, Takai H, de LT (2012) Telomeric 3′ overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST. Cell 150:39–52CrossRefGoogle Scholar
  36. 36.
    Modrich P (2006) Mechanisms in eukaryotic mismatch repair. J Biol Chem 281:30305–30309CrossRefGoogle Scholar
  37. 37.
    Pasero P, Vindigni A (2017) Nucleases acting at stalled forks: how to reboot the replication program with a few shortcuts. Annu Rev Genet 51:477–499CrossRefGoogle Scholar
  38. 38.
    Orans J, McSweeney EA, Iyer RR et al (2011) Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family. Cell 145:212–223CrossRefGoogle Scholar
  39. 39.
    Shi Y, Hellinga HW, Beese LS (2017) Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I. Proc Natl Acad Sci U S A 114(23):6010–6015CrossRefGoogle Scholar
  40. 40.
    Yang S-H, Zhou R, Campbell J et al (2013) The SOSS1 single-stranded DNA binding complex promotes DNA end resection in concert with Exo1. EMBO J 32:126–139CrossRefGoogle Scholar
  41. 41.
    Genschel J, Modrich P (2003) Mechanism of 5′-directed excision in human mismatch repair. Mol Cell 12:1077–1086CrossRefGoogle Scholar
  42. 42.
    Finkelstein IJ, Visnapuu M-L, Greene EC (2010) Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 468:983–987CrossRefGoogle Scholar
  43. 43.
    Duffy S, Tsao KL, Waugh DS (1998) Site-specific, enzymatic biotinylation of recombinant proteins in Spodoptera frugiperda cells using biotin acceptor peptides. Anal Biochem 262:122–128CrossRefGoogle Scholar
  44. 44.
    Sorenson AE, Askin SP, Schaeffer PM (2015) In-gel detection of biotin–protein conjugates with a green fluorescent streptavidin probe. Anal Methods 7:2087–2092CrossRefGoogle Scholar
  45. 45.
    Soniat MM, Myler LR, Schaub JM et al (2017) Next-Generation DNA curtains for single-molecule studies of homologous recombination. In: Eichman BF (ed) Methods in enzymology. Academic Press, Cambridge, MA, pp 259–281Google Scholar
  46. 46.
    Finkelstein IJ, Greene EC (2011) Supported lipid bilayers and DNA curtains for high-throughput single-molecule studies. Methods Mol Biol 745:447–461CrossRefGoogle Scholar
  47. 47.
    Miller AS, Daley JM, Pham NT et al (2017) A novel role of the Dna2 translocase function in DNA break resection. Genes Dev 31:503–510CrossRefGoogle Scholar
  48. 48.
    Zhou C, Pourmal S, Pavletich NP (2015) Dna2 nuclease-helicase structure, mechanism and regulation by Rpa. elife 4:e09832CrossRefGoogle Scholar
  49. 49.
    Masuda-Sasa T, Imamura O, Campbell JL (2006) Biochemical analysis of human Dna2. Nucleic Acids Res 34:1865–1875CrossRefGoogle Scholar
  50. 50.
    Croteau DL, Popuri V, Opresko PL et al (2014) Human RecQ helicases in DNA repair, recombination, and replication. Annu Rev Biochem 83:519–552CrossRefGoogle Scholar
  51. 51.
    Bernstein KA, Gangloff S, Rothstein R (2010) The RecQ DNA helicases in DNA repair. Annu Rev Genet 44:393–417CrossRefGoogle Scholar
  52. 52.
    Beresten SF, Stan R, van Brabant AJ et al (1999) Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes. Protein Expr Purif 17:239–248CrossRefGoogle Scholar
  53. 53.
    Janscak P, Garcia PL, Hamburger F et al (2003) Characterization and mutational analysis of the RecQ core of the bloom syndrome protein. J Mol Biol 330:29–42CrossRefGoogle Scholar
  54. 54.
    Kitano K (2014) Structural mechanisms of human RecQ helicases WRN and BLM. Front Genet 5:366CrossRefGoogle Scholar
  55. 55.
    Bernstein DA, Keck JL (2003) Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family. Nucleic Acids Res 31:2778–2785CrossRefGoogle Scholar
  56. 56.
    Kim YM, Choi B-S (2010) Structure and function of the regulatory HRDC domain from human Bloom syndrome protein. Nucleic Acids Res 38:7764–7777CrossRefGoogle Scholar
  57. 57.
    Liu Z, Macias MJ, Bottomley MJ et al (1999) The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins. Structure 7:1557–1566CrossRefGoogle Scholar
  58. 58.
    Brosh RM, Li J-L, Kenny MK et al (2000) Replication protein A physically interacts with the Bloom’s syndrome protein and stimulates its helicase activity. J Biol Chem 275:23500–23508CrossRefGoogle Scholar
  59. 59.
    Doherty KM, Sommers JA, Gray MD et al (2005) Physical and functional mapping of the replication protein A interaction domain of the Werner and Bloom syndrome helicases. J Biol Chem 280:29494–29505CrossRefGoogle Scholar
  60. 60.
    Kang D, Lee S, Ryu K-S et al (2018) Interaction of replication protein A with two acidic peptides from human Bloom syndrome protein. FEBS Lett 592:547–558CrossRefGoogle Scholar
  61. 61.
    Guo R-B, Rigolet P, Ren H et al (2007) Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein. Nucleic Acids Res 35:6297–6310CrossRefGoogle Scholar
  62. 62.
    Chatterjee S, Zagelbaum J, Savitsky P et al (2014) Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures. Nat Commun 5:ncomms6556CrossRefGoogle Scholar
  63. 63.
    Yodh JG, Stevens BC, Kanagaraj R et al (2009) BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation. EMBO J 28:405–416CrossRefGoogle Scholar
  64. 64.
    Newman JA, Savitsky P, Allerston CK et al (2015) Crystal structure of the Bloom’s syndrome helicase indicates a role for the HRDC domain in conformational changes. Nucleic Acids Res 43:5221–5235CrossRefGoogle Scholar
  65. 65.
    Swan MK, Legris V, Tanner A et al (2014) Structure of human Bloom’s syndrome helicase in complex with ADP and duplex DNA. Acta Crystallogr D Biol Crystallogr 70:1465–1475CrossRefGoogle Scholar
  66. 66.
    Nguyen GH, Dexheimer TS, Rosenthal AS et al (2013) A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem Biol 20:55–62CrossRefGoogle Scholar
  67. 67.
    Karow JK, Chakraverty RK, Hickson ID (1997) The Bloom’s syndrome gene product is a 3′–5′ DNA helicase. J Biol Chem 272:30611–30614CrossRefGoogle Scholar
  68. 68.
    Yao J, Larson DR, Vishwasrao HD et al (2005) Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution. Proc Natl Acad Sci U S A 102:14284–14289CrossRefGoogle Scholar
  69. 69.
    Ebenstein Y, Mokari T, Banin U (2002) Fluorescence quantum yield of CdSe/ZnS nanocrystals investigated by correlated atomic-force and single-particle fluorescence microscopy. Appl Phys Lett 80:4033–4035CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Michael M. Soniat
    • 1
  • Logan R. Myler
    • 1
  • Ilya J. Finkelstein
    • 1
    • 2
    Email author
  1. 1.Department of Molecular Biosciences and Institute for Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA
  2. 2.Center for Systems and Synthetic BiologyThe University of Texas at AustinAustinUSA

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