DNA Nanotechnology pp 235-254

Part of the Methods in Molecular Biology book series (MIMB, volume 749)

DNA Contour Length Measurements as a Tool for the Structural Analysis of DNA and Nucleoprotein Complexes

Protocol

Abstract

The atomic force microscope (AFM) is a widely used tool to image DNA and nucleoprotein complexes at the molecular level. This is because the AFM is relatively easy to operate, has the capability to image biomolecules under aqueous solutions, and, most importantly, can image mesoscopic macromolecular structures that are too complex to be studied by X-ray or NMR and too small to be visualized with the optical microscope. Although there are many AFM studies about the structure and the physical properties of DNA, only in few cases a rigorous method has been applied to analyze AFM images. This chapter describes procedures to prepare DNA and nucleoprotein complexes for AFM imaging and methods used to carry out simple image measurements to obtain structural data. In particular, methods to measure DNA contour length and the volume of free or DNA-bound proteins are presented and discussed.

Key words

Atomic force microscopy Image analysis Contour length Volume DNA Nucleoprotein complex 

References

  1. 1.
    Bustamante, C, Rivetti, C (1996). Visualizing protein-nucleic acid interactions on a large scale with the scanning force microscope. Ann. Rev. Biophys. Biomol. Struc. 25, 395–429.CrossRefGoogle Scholar
  2. 2.
    Rivetti, C, Guthold, M, Bustamante, C (1996). Scanning Force Microscopy of DNA deposited on mica: Equilibration versus kinetic trapping studied by polymer chain analysis. J. Mol. Biol. 264, 919–932.CrossRefGoogle Scholar
  3. 3.
    Rivetti, C, Walker, C, Bustamante, C (1998). Polymer chain statistics and conformational analysis of DNA molecules with bends or sections of different flexibility. J. Mol. Biol. 280, 41–59.CrossRefGoogle Scholar
  4. 4.
    Zuccheri, G, Scipioni, A, Cavaliere, V, Gargiulo, G, De Santis, P, Samori, B (2001). Mapping the intrinsic curvature and flexibility along the DNA chain. Proc. Natl. Acad. Sci. USA. 98, 3074–3079.CrossRefGoogle Scholar
  5. 5.
    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.CrossRefGoogle Scholar
  6. 6.
    Moreno-Herrero, F, Herrero, P, Colchero, J, Baro, AM, Moreno, F (2001). Imaging and mapping protein-binding sites on DNA regulatory regions with atomic force microscopy. Biochem. Biophys. Res. Commun. 280, 151–157.CrossRefGoogle Scholar
  7. 7.
    Folli, C, Mangiarotti, L, Folloni, S, Alfieri, B, Gobbo, M, Berni, R et al. (2008). Specificity of the TraA-DNA interaction in the regulation of the pPD1-encoded sex pheromone response in Enterococcus faecalis. J. Mol. Biol. 380, 932–945.CrossRefGoogle Scholar
  8. 8.
    Rivetti, C, Guthold, M, Bustamante, C (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J. 18, 4464–4475.CrossRefGoogle Scholar
  9. 9.
    Verhoeven, EE, Wyman, C, Moolenaar, GF, Hoeijmakers, JH, Goosen, N (2001). Archi­tecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition. EMBO J. 20, 601–611.CrossRefGoogle Scholar
  10. 10.
    Rivetti, C, Codeluppi, S, Dieci, G, Bustamante, C (2003). Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases. J. Mol. Biol. 326, 1413–1426.CrossRefGoogle Scholar
  11. 11.
    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.CrossRefGoogle Scholar
  12. 12.
    Cellai, S, Mangiarotti, L, Vannini, N, Naryshkin, N, Kortkhonjia, E, Ebright, RH et al. (2007). Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex. EMBO Rep. 8, 271–278.CrossRefGoogle Scholar
  13. 13.
    Mangiarotti, L, Cellai, S, Ross, W, Bustamante, C, Rivetti, C (2009). Sequence-dependent upstream DNA-RNA polymerase interactions in the open complex with lambdaPR and lambdaPRM ­promoters and implications for the mechanism of promoter interference. J. Mol. Biol. 385, 748–760.CrossRefGoogle Scholar
  14. 14.
    Sushko, ML, Shluger, AL, Rivetti, C (2006). Simple model for DNA adsorption onto a mica surface in 1:1 and 2:1 electrolyte solutions. Langmuir 22, 7678–7688.CrossRefGoogle Scholar
  15. 15.
    Lyubchenko, YL, Shlyakhtenko, LS, Harrington, RE, Oden, PI, Lindsay, SM (1993). Atomic force microscopy of long DNA: Imaging in air and under water. Proc. Natl. Acad. Sci. USA. 90, 2137–2140.CrossRefGoogle Scholar
  16. 16.
    Bussiek, M, Mucke, N, Langowski, J (2003). Polylysine-coated mica can be used to observe systematic changes in the supercoiled DNA conformation by scanning force microscopy in solution. Nucleic Acids Res. 31, e137.CrossRefGoogle Scholar
  17. 17.
    Podesta, A, Imperadori, L, Colnaghi, W, Finzi, L, Milani, P, Dunlap, D (2004). Atomic force microscopy study of DNA deposited on poly L-ornithine-coated mica. J. Microsc. 215, 236–240.CrossRefGoogle Scholar
  18. 18.
    Bustamante, C, Rivetti, C, Keller, DJ (1997). Scanning Force Microscopy under aqueous solution. Current Opinion in Structural Biology 7, 709–716.CrossRefGoogle Scholar
  19. 19.
    Ficarra, E, Benini, L, Macii, E, Zuccheri, G (2005). Automated DNA fragments recognition and sizing through AFM image processing. IEEE Trans. Inf. Technol. Biomed. 9, 508–517.CrossRefGoogle Scholar
  20. 20.
    Marek, J, Demjenova, E, Tomori, Z, Janacek, J, Zolotova, I, Valle, F et al. (2005). Interactive measurement and characterization of DNA molecules by analysis of AFM images. Cytometry A. 63, 87–93.Google Scholar
  21. 21.
    Rivetti, C (2009). A simple and optimized length estimator for digitized DNA contours. Cytometry A. 75A, 854–861.Google Scholar
  22. 22.
    Tang, M, Cecconi, C, Bustamante, C, Rio, DC (2007). Analysis of P element transposase protein-DNA interactions during the early stages of transposition. J. Biol. Chem. 282, 29002–29012.CrossRefGoogle Scholar
  23. 23.
    Minh, PN, Devroede, N, Massant, J, Maes, D, Charlier, D (2009). Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy. Nucleic Acids Res. 37, 1463–1476.CrossRefGoogle Scholar
  24. 24.
    Russ, J. C. (1995) The Image Processing Handbook. 2nd Ed. CRC, London.Google Scholar
  25. 25.
    Wyman, C, Grotkopp, E, Bustamante, C, Nelson, HCM (1995). Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy. EMBO J. 14, 117–123.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of ParmaParmaItaly

Personalised recommendations