Analysis of DNA–Protein Complexes by Atomic Force Microscopy Imaging: The Case of TRF2–Telomeric DNA Wrapping

  • Sabrina PisanoEmail author
  • Eric Gilson
Part of the Methods in Molecular Biology book series (MIMB, volume 1886)


Atomic force microscopy (AFM) is a non-optical microscopy that enables the acquisition at the nanoscale level of a 3D topographical image of the sample. For 30 years, AFM has been a valuable tool in life sciences to study biological samples in the field of tissue, cellular and molecular imaging, of mechanical properties and of force spectroscopy. Since the early beginnings of the technique, AFM has been extensively exploited as an imaging tool for structural studies of nucleic acids and nucleoprotein complexes. The morphometric analysis performed on the images can unveil specific structural and functional aspects of the sample, such as the multimerization state of proteins bound to DNA, or DNA conformational changes led by the DNA-binding proteins. Herein, a method for analyzing a complex formed by a telomeric DNA sequence wrapped around the TRF2 binding protein is presented. The described procedure could be applied to the study of any type of DNA–protein complex.

Key words

Atomic force microscopy DNA–protein complex Volume analysis Contour length Telomeric DNA TRF2 Wrapping 



This work was funded by the Ligue Nationale contre le Cancer (Equipe labélisée) and Investments for the Future LABEXSIGNALIFE (reference ANR-236 11-LABX-0028-01).

The authors acknowledge PICMI, the IRCAN’s Imaging core facility. The atomic force microscopy of PICMI was supported by the Association pour la Recherche sur le Cancer (ARC), by the Infrastructures en Biologie Santé et Agronomie (IBiSA) and by the Conseil General 06 de la Région Provence Alpes-Côte.


  1. 1.
    Binnig G, Rohrer H, Gerber C, Weibel E (1982) Surface studies by scanning tunneling microscopy. Phys Rev Lett 49:57–61. Scholar
  2. 2.
    Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56:930–933. Scholar
  3. 3.
    Allison DP, Mortensen NP, Sullivan CJ, Doktycz MJ (2010) Atomic force microscopy of biological samples. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2:618–634. Scholar
  4. 4.
    Ozkan AD, Topal AE, Dana A et al (2016) Atomic force microscopy for the investigation of molecular and cellular behavior. Micron 89:60–76. Scholar
  5. 5.
    Wang H, Nora GJ, Ghodke H, Opresko PL (2011) Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding. J Biol Chem 286:7479–7489. Scholar
  6. 6.
    Li BS, Wei B, Goh MC ((2012)) Direct visualization of the formation of RecA/dsDNA complexes at the single-molecule level. Micron 43:1073–1075. Scholar
  7. 7.
    Poulet A, Pisano S, Faivre-Moskalenko C et al (2012) The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA. Nucleic Acids Res 40:2566–2576. Scholar
  8. 8.
    Shlyakhtenko LS, Lushnikov AY, Miyagi A, Lyubchenko YL (2012) Specificity of binding of single-stranded DNA-binding protein to its target. Biochemistry (Mosc) 51:1500–1509. Scholar
  9. 9.
    Buechner CN, Maiti A, Drohat AC, Tessmer I (2015) Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging. Nucleic Acids Res 43:2716–2729. Scholar
  10. 10.
    Jiang Y, Marszalek PE (2011) Atomic force microscopy captures MutS tetramers initiating DNA mismatch repair. EMBO J 30:2881–2893. Scholar
  11. 11.
    Cicconi A, Micheli E, Vernì F et al (2017) The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response. Nucleic Acids Res 45:3068–3085. Scholar
  12. 12.
    Benarroch-Popivker D, Pisano S, Mendez-Bermudez A et al (2016) TRF2-mediated control of telomere DNA topology as a mechanism for chromosome-end protection. Mol Cell 61:274–286. Scholar
  13. 13.
    Pisano S, Pascucci E, Cacchione S et al (2006) AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning. Biophys Chem 124:81–89. Scholar
  14. 14.
    Fu H, Freedman BS, Lim CT et al (2011) Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract. Chromosoma 120:245–254. Scholar
  15. 15.
    North JA, Šimon M, Ferdinand MB et al (2014) Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure. Nucleic Acids Res 42:4922–4933. Scholar
  16. 16.
    Athwal RK, Walkiewicz MP, Baek S et al (2015) CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells. Epigenetics Chromatin 8:2. Scholar
  17. 17.
    Kasas S, Thomson NH, Smith BL et al (1997) Escherichia coli RNA polymerase activity observed using atomic force microscopy. Biochemistry (Mosc) 36:461–468. Scholar
  18. 18.
    Wang H, Bash R, Yodh JG et al (2002) Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin. Biophys J 83:3619–3625. Scholar
  19. 19.
    Wang H, Bash R, Yodh JG et al (2004) Using atomic force microscopy to study nucleosome remodeling on individual nucleosomal arrays in situ. Biophys J 87:1964–1971. Scholar
  20. 20.
    Shlyakhtenko LS, Lushnikov AY, Lyubchenko YL (2009) Dynamics of nucleosomes revealed by time-lapse atomic force microscopy. Biochemistry (Mosc) 48:7842–7848. Scholar
  21. 21.
    Pisano S, Marchioni E, Galati A et al (2007) Telomeric nucleosomes are intrinsically mobile. J Mol Biol 369:1153–1162. Scholar
  22. 22.
    Pisano S, Leoni D, Galati A et al (2010) The human telomeric protein hTRF1 induces telomere-specific nucleosome mobility. Nucleic Acids Res 38:2247–2255. Scholar
  23. 23.
    Montel F, Castelnovo M, Menoni H et al (2011) RSC remodeling of oligo-nucleosomes: an atomic force microscopy study. Nucleic Acids Res 39:2571–2579. Scholar
  24. 24.
    Yeeles JTP, van Aelst K, Dillingham MS, Moreno-Herrero F (2011) Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease. Mol Cell 42:806–816. Scholar
  25. 25.
    Hansma HG, Revenko I, Kim K, Laney DE (1996) Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids. Nucleic Acids Res 24:713–720. Scholar
  26. 26.
    Rivetti C, Guthold M, Bustamante C (1996) Scanning force microscopy of DNA deposited onto mica: equilibration versus kinetic trapping studied by statistical polymer chain analysis. J Mol Biol 264:919–932. Scholar
  27. 27.
    Pastré D, Hamon L, Landousy F et al (2006) Anionic polyelectrolyte adsorption on mica mediated by multivalent cations: a solution to DNA imaging by atomic force microscopy under high ionic strengths. Langmuir 22:6651–6660. Scholar
  28. 28.
    Voigtländer B (2015) Amplitude modulation (AM) mode in dynamic atomic force microscopy. In: Scanning probe microscopy, atomic force microscopy and scanning tunneling microscopy, vol 69. Springer-Verlag, Berlin Heidelberg, pp 187–204. Scholar
  29. 29.
    Lyubchenko YL, Shlyakhtenko LS (2016) Imaging of DNA and protein-DNA complexes with atomic force microscopy. Crit Rev Eukaryot Gene Expr 26:63–96. Scholar
  30. 30.
    Verhoeven EE, Wyman C, Moolenaar GF et al (2001) Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition. EMBO J 20:601–611. Scholar
  31. 31.
    Rivetti C, Codeluppi S (2001) Accurate length determination of DNA molecules visualized by atomic force microscopy: evidence for a partial B- to A-form transition on mica. Ultramicroscopy 87:55–66. Scholar
  32. 32.
    García R, Pérez R (2002) Dynamic atomic force microscopy methods. Surf Sci Rep 47:197–301. Scholar
  33. 33.
    Moreno-Herrero F, Colchero J, Baró AM (2003) DNA height in scanning force microscopy. Ultramicroscopy 96:167–174. Scholar
  34. 34.
    Santos S, Barcons V, Christenson HK et al (2011) The intrinsic resolution limit in the atomic force microscope: implications for heights of nano-scale features. PLoS One 6:e23821. Scholar
  35. 35.
    Bustamante C, Keller D, Yang G (1993) Scanning force microscopy of nucleic acids and nucleoprotein assemblies. Curr Opin Struct Biol 3:363–372. Scholar
  36. 36.
    Schneider SW, Lärmer J, Henderson RM, Oberleithner H (1998) Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pflugers Arch 435:362–367. Scholar
  37. 37.
    Nettikadan S, Tokumasu F, Takeyasu K (1996) Quantitative analysis of the transcription factor AP2 binding to DNA by atomic force microscopy. Biochem Biophys Res Commun 226:645–649. Scholar
  38. 38.
    Broccoli D, Smogorzewska A, Chong L, de Lange T (1997) Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat Genet 17:231–235. Scholar
  39. 39.
    Luijsterburg MS, White MF, van Driel R, Dame RT (2008) The major architects of chromatin: architectural proteins in bacteria, archaea and eukaryotes. Crit Rev Biochem Mol Biol 43:393–418. Scholar
  40. 40.
    Rivetti C, Guthold M, Bustamante C (1999) Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J 18:4464–4475. Scholar
  41. 41.
    Beerens N, Hoeijmakers JHJ, Kanaar R et al (2005) The CSB protein actively wraps DNA. J Biol Chem 280:4722–4729. Scholar
  42. 42.
    Wang H, Dodd IB, Dunlap DD et al (2013) Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor. Nucleic Acids Res 41:5746–5756. Scholar
  43. 43.
    Cary RB, Peterson SR, Wang J et al (1997) DNA looping by Ku and the DNA-dependent protein kinase. Proc Natl Acad Sci U S A 94:4267–4272. Scholar
  44. 44.
    Schnitzler GR, Cheung CL, Hafner JH et al (2001) Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips. Mol Cell Biol 21:8504–8511. Scholar
  45. 45.
    Tahirov TH, Sato K, Ichikawa-Iwata E et al (2002) Mechanism of c-Myb-C/EBP beta cooperation from separated sites on a promoter. Cell 108:57–70. Scholar
  46. 46.
    Bussiek M, Tóth K, Brun N, Langowski J (2005) DNA-loop formation on nucleosomes shown by in situ scanning force microscopy of supercoiled DNA. J Mol Biol 345:695–706. Scholar
  47. 47.
    Lia G, Indrieri M, Owen-Hughes T et al (2008) ATP-dependent looping of DNA by ISWI. J Biophotonics 1:280–286. Scholar
  48. 48.
    Neaves KJ, Cooper LP, White JH et al (2009) Atomic force microscopy of the EcoKI type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping. Nucleic Acids Res 37:2053–2063. Scholar
  49. 49.
    Wu D, Kaur P, Li ZM et al (2016) Visualizing the path of DNA through proteins using DREEM imaging. Mol Cell 61:315–323. Scholar
  50. 50.
    Ramanujan S (1914) Modular equations and approximations to π. Quart J Pure App Math 45:350–372Google Scholar
  51. 51.
    Shlyakhtenko LS, Gall AA, Lyubchenko YL (2013) Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy. Methods Mol Biol 931:295–312. Scholar
  52. 52.
    Heddle JG, Mitelheiser S, Maxwell A, Thomson NH (2004) Nucleotide binding to DNA gyrase causes loss of DNA wrap. J Mol Biol 337:597–610. Scholar
  53. 53.
    Ratcliff GC, Erie DA (2001) A novel single-molecule study to determine protein–protein association constants. J Am Chem Soc 123:5632–5635. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Université Côte d’Azur, CNRS UMR 7284/INSERM U108, Institute for Research on Cancer and Aging, Nice (IRCAN), Medical SchoolNiceFrance
  2. 2.International Laboratory in Hematology and Cancer, Pôle Sino-Français de Recherche en Sciences du Vivant et Génomique, Shanghai Ruijin HospitalShanghai Jiao Tong University School of Medicine/Ruijin Hospital/CNRS/INSERM/Nice UniversityShanghaiChina
  3. 3.Department of Genetics, CHU NiceUniversité Côte d’AzurNiceFrance

Personalised recommendations