Abstract
Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate, and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nanometer resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA-bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA, and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.
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References
Janicijevic, A., Ristic, D. and Wyman, C. (2003) The molecular machines of DNA repair: scanning force microscopy analysis of their architecture. J Microsc. 212, 264–72.
Dame, R.T., Wyman, C. and Goosen, N. (2003) Insights into the regulation of transcription by scanning force microscopy. J Microsc. 212, 244–53.
Erie, D.A., Yang, G., Schultz, H. C. and Bustamante, C. (1994) DNA bending by Cro protein in specific and nonspecific complexes: implications for protein site recognition and specificity. Science 266, 1562–6.
Ristic, D., Modesti, M., van der Heijden, T., van Noort, J., Dekker, C., Kanaar, R. and Wyman, C. (2005) Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. 33, 3292–302.
Bustamante, C., Vesenka, J., Tang, C. L., Rees, W., Guthold, M. and Keller, R. (1992) Circular DNA molecules imaged in air by scanning force microscopy. Biochemistry 31, 22–6.
Vesenka, J., Guthold, M., Tang, C. L., Keller, D., Delaine, E. and Bustamante, C. (1992) Substrate preparation for reliable imaging of DNA molecules with the scanning force microscope. Ultramicroscopy 42–44, 1243–9.
Hansma, H.G. and Laney, D.E. (1996) DNA binding to mica correlates with cationic radius: assay by atomic force microscopy. Biophys J. 70, 1933–9.
Han, W., Lindsay, S. M., Dlakic, M. and Harrington, R. E. (1997) Kinked DNA. Nature 386, 563.
Rivetti, C., Guthold, M. and 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, 919–32.
Beerens, N., Hoeijmakers, J. H., Kanaar, R., Vermeulen, W. and Wyman, C. (2005) The CSB protein actively wraps DNA. J Biol Chem. 280, 4722–9.
Ratcliff, G.C. and Erie, D.A. (2001) A novel single-molecule study to determine protein--protein association constants. J Am Chem Soc. 123, 5632–5.
Wyman, C., Rombel, I., North, A. K., Bustamante, C. and Kustu, S. (1997) Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science 275, 1658–61.
van der Linden, E., Sanchez, H., Kinoshita, E., Kanaar, R. and Wyman, C. (2009) RAD50 and NBS1 form a stable complex functional in DNA binding and tethering. Nucleic Acids Res. 37, 1580–8.
Janicijevic, A., Sugasawa, K., Shimizu, Y., Hanaoka, F., Wijgers, N., Djurica, M., Hoeijmakers, J. H. and Wyman, C. (2003) DNA bending by the human damage recognition complex XPC-HR23B. DNA Repair (Amst). 2, 325–36.
Dame, R.T., van Mameren, J., Luijsterburg, M. S., Mysiak, M. E., Janicijevic, A., Pazdzior, G., van der Vliet, P. C., Wyman, C. and Wuite, G. J. (2005) Analysis of scanning force microscopy images of protein-induced DNA bending using simulations. Nucleic Acids Res. 33, e68.
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Ristic, D., Sanchez, H., Wyman, C. (2011). Sample Preparation for SFM Imaging of DNA, Proteins, and DNA–Protein Complexes. In: Peterman, E., Wuite, G. (eds) Single Molecule Analysis. Methods in Molecular Biology, vol 783. Humana Press. https://doi.org/10.1007/978-1-61779-282-3_12
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DOI: https://doi.org/10.1007/978-1-61779-282-3_12
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