Summary
Protein–DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein–DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual Protein–DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of Protein–DNA interactions.
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References
Hillisch, A., Lorenz, M., and Diekmann, S. (2001) Recent advances in FRET: distance determination in Protein–DNA complexes. Curr. Opinion Struct. Biol. 11, 201–207.
Holbrook, S.R. (2005) RNA structure: the long and the short of it. Curr. Opinion Struct. Biol. 15, 302–308.
Yan, Y., and Marriott, G. (2003) Analysis of protein interactions using fluorescence technologies. Curr. Opinion Chem. Biol. 7, 635–640.
Michalet, X., Kapanidis, A.N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M., and Weiss, S. (2003) The power and prospects of fluorescence microscopies and spectroscopies. Annu. Rev. Biophys. Biomol. Struct. 32, 161–82.
Lorenz, M., Hillisch, A., Payet, D., Buttinelli, M., Travers, A., and Diekmann, S. (1999) DNA bending induced by high mobility group proteins studied by fluorescence resonance energy transfer. Biochemistry 38, 12150–12158.
Selvin, P.R. (2000) The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7, 730–734.
Stuhmeier, F., Hillisch, A., Clegg, R.M., and Diekman, S. (2000) Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP. J. Mol. Biol. 302, 1081–1100.
Clegg, R.M. (1992) Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211, 353–388.
Stryer, L., and Haugland, R.P. (1967) Energy transfer: a spectroscopic ruler. Proc. Natl Acad. Sci. USA 58, 719–726.
Stuhmeier, F., Hillisch, A., Clegg, R.M., and Diekman, S. (2000) Practical aspects of fluorescence resonance energy transfer (FRET) and its applications in nucleic acid biochemistry. DNA–Protein Interactions. Edited by Travers A., Buckle, M., Oxford: Oxford University Press, 77–94.
Bera, A., Roche, A. C., and Nandi, P. K. (2007) Bending and unwinding of nucleic acid by prion protein. Biochemistry 46, 1320–1328.
Lorenz, M., and Diekmann, S. (2006) Distance determination in Protein–DNA complexes using fluorescence resonance energy transfer. Methods Mol. Biol. 335, 243–255.
Passner, J. M., and Steitz, T. A. (1997) The structure of a CAP–DNA complex having two cAMP molecules bound to each monomer. Proc. Natl Acad. Sci. USA 94, 2843–2847.
Hieb, A. R., Halsey, W. A., Betterton, M. D., Perkins, T. T., Kugel, J. F., and Goodrich, J. A. (2007) TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability. J. Mol. Biol. 372, 619–632.
Dragan, A. I., Klass, J., Read, C., Churchill, M. E., Crane-Robinson, C., and Privalov, P. L. (2003) DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution. J. Mol. Biol. 331, 795–813.
Kuznetsov, S. V., Sugimura, S., Vivas, P., Crothers, D. M., and Ansari, A. (2006) Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF. Proc. Natl Acad. Sci. USA 103, 18515–18520.
Lorenz, M., Hillisch, A., Goodman, S. D., and Diekmann, S. (1999) Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer. Nucleic Acids Res. 27, 4619–4625.
Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., and Tainer, J. A. (2004) Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair. Cell 116, 39–50.
Xiao, J., and Singleton, S. F. (2002) Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer. J. Mol. Biol. 320, 529–558.
McKinney, S. A., Joo, C., and Ha, T. (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys. J. 91, 1941–1951.
Gupta, R. C., Golub, E. I., Wold, M. S., and Radding, C. M. (1998) Polarity of DNA strand exchange promoted by recombination proteins of the RecA family. Proc. Natl Acad. Sci. USA 95, 9843–9848.
Kuznetsov, S. V., Kozlov, A. G., Lohman, T. M., and Ansari, A. (2006) Microsecond dynamics of Protein–DNA interactions: direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer. J. Mol. Biol. 359, 55–65.
Lucius, A. L., Jason Wong, C., and Lohman, T. M. (2004) Fluorescence stopped-flow studies of single turnover kinetics of E. coli RecBCD helicase-catalyzed DNA unwinding. J. Mol. Biol. 339, 731–750.
Kvaratskhelia, M., Wardleworth, B. N., Bond, C. S., Fogg, J. M., Lilley, D. M., and White, M. F. (2002) Holliday junction resolution is modulated by archaeal chromatin components in vitro. J. Biol. Chem. 277, 2992–2996.
Furey, W. S., Joyce, C. M., Osborne, M. A., Klenerman, D., Peliska, J. A., and Balasubramanian, S. (1998) Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment. Biochemistry 37, 2979–2990.
Mukhopadhyay, J., Mekler, V., Kortkhonjia, E., Kapanidis, A. N., Ebright, Y. W., and Ebright, R. H. (2003) Fluorescence resonance energy transfer (FRET) in analysis of transcription-complex structure and function. Methods Enzymol. 371, 144–159.
Heyduk, T., and Niedziela-Majka, A. (2001) Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes. Biopolymers 61, 201–213.
Margeat, E., Kapanidis, A. N., Tinnefeld, P., Wang, Y., Mukhopadhyay, J., Ebright, R. H., and Weiss, S. (2006) Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes. Biophys. J. 90, 1419–1431.
Kapanidis, A. N., Margeat, E., Ho, S. O., Kortkhonjia, E., Weiss, S., and Ebright, R. H. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147.
Lee, S. P., and Han, M. K. (1997) Fluorescence assays for DNA cleavage. Methods Enzymol. 278, 343–363.
Eggeling, C., Jager, S., Winkler, D., and Kask, P. (2005) Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction. Curr. Pharm. Biotechnol. 6, 351–371.
Ghosh, S. S., Eis, P. S., Blumeyer, K., Fearon, K., and Millar, D. P. (1994) Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer. Nucleic Acids Res. 22, 3155–3159.
Hiller, D. A., Rodriguez, A. M., and Perona, J. J. (2005) Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC. J. Mol. Biol. 354, 121–136.
Ray, P. C., Fortner, A., and Darbha, G. K. (2006) Gold nanoparticle based FRET assay for the detection of DNA cleavage. J Phys Chem B 110, 20745–20748.
Lin, J., Gao, H., Schallhorn, K. A., Harris, R. M., Cao, W., and Ke, P. C. (2007) Lesion recognition and cleavage by endonuclease V: a single-molecule study. Biochemistry 46, 7132–7137.
van der Meer, B. W. (2002) Kappa-squared: from nuisance to new sense. J. Biotechnol. 82, 181–196.
Klostermeier, D., and Millar, D. P. (2001) Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids. Biopolymers 61, 159–179.
Cornish, P. V., and Ha, T. (2007) A survey of single-molecule techniques in chemical biology. ACS Chem. Biol. 2, 53–61.
Ha, T. (2001) Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86.
Ha, T. (2004) Structural dynamics and processing of nucleic acids revealed by single-molecule spectroscopy. Biochemistry 43, 4055–4063.
Ritort, F. (2006) Single-molecule experiments in biological physics: methods and applications J. Phys.: Condens. Matter 18, R531–R583.
Moerner, W. E., and Fromm, D. P. (2003) Methods of single-molecule fluorescence spectroscopy and microscopy. Rev. Sci. Instrum. 74, 3597.
Haustein, E., and Schwille, P. (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annu. Rev. Biophys. Biomol. Struct. 36, 151–169.
Schwille, P. (2003) TIR-FCS: staying on the surface can sometimes be better. Biophys. J. 85, 2783–2784.
Wazawa, T., and Ueda, M. (2005) Total internal reflection fluorescence microscopy in single molecule nanobioscience. Adv. Biochem. Eng. Biotechnol. 95, 77–106.
Rasnik, I., McKinney, S. A., and Ha, T. (2005) Surfaces and orientations: much to FRET about? Acc. Chem. Res. 38, 542–548.
Cisse, I., Okumus, B., Joo, C., and Ha, T. (2007) Fueling protein DNA interactions inside porous nanocontainers. Proc. Natl Acad. Sci. USA 104, 12646–12650.
Myong, S., Bruno, M. M., Pyle, A. M., and Ha, T. (2007) Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317, 513–516.
Lu, H. P., Iakoucheva, L. M., and Ackerman, E. J. (2001) Single-molecule conformational dynamics of fluctuating noncovalent DNA–protein interactions in DNA damage recognition. J. Am. Chem. Soc. 123, 9184–9185.
Segers-Nolten, G. M., Wyman, C., Wijgers, N., Vermeulen, W., Lenferink, A. T., Hoeijmakers, J. H., Greve, J., and Otto, C. (2002) Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes. Nucleic Acids Res. 30, 4720–4727.
Lemay, J. F., Penedo, J. C., Tremblay, R., Lilley, D. M., and Lafontaine, D. A. (2006) Folding of the adenine riboswitch. Chem. Biol. 13, 857–868.
Braslavsky, I., Hebert, B., Kartalov, E., and Quake, S. R. (2003) Sequence information can be obtained from single DNA molecules. Proc. Natl Acad. Sci. USA 100, 3960–3964.
Groll, J., Amirgoulova, E. V., Ameringer, T., Heyes, C. D., Rocker, C., Nienhaus, G. U., and Moller, M. (2004) Biofunctionalized, ultrathin coatings of cross-linked star-shaped poly(ethylene oxide) allow reversible folding of immobilized proteins. J. Am. Chem. Soc. 126, 4234–4239.
Adachi, K., Yasuda, R., Noji, H., Itoh, H., Harada, Y., Yoshida, M., and Kinosita, K., Jr. (2000) Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging. Proc. Natl Acad. Sci. USA 97, 7243–7247.
Kastner, C. N., Prummer, M., Sick, B., Renn, A., Wild, U. P., and Dimroth, P. (2003) The citrate carrier CitS probed by single-molecule fluorescence spectroscopy. Biophys. J. 84, 1651–1659.
Boukobza, E., Sonnenfeld, A., and Haran, G. (2001) Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy. J. Phys. Chem. B 105, 12165–12170.
Kapanidis, A. N., and Weiss, S. (2002) Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J. Chem. Phys. 117, 10953–10964.
Sapsford, K. E., Berti, L., and Medintz, I. L. (2006) Materials for fluorescence resonance energy transfer analysis: beyond traditional donor–acceptor combinations. Angew. Chem. Int. Ed. Engl. 45, 4562–4589.
Acknowledgments
We thank the Biological and Biotechnology Science Research Council (UK), the Royal Society (UK), and the National Sciences and Engineering Research Council (Canada), and the Universities of Sherbrooke (Canada) and St Andrews (UK) for financial support. We also thank all members of our labs for helpful discussion and critical reading of the manuscript. J. C. P is a Fellow of the Scottish Universities Physics Alliance (SUPA). DAL is a CIHR New Investigator scholar.
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Blouin, S., Craggs, T.D., Lafontaine, D.A., Penedo, J.C. (2009). Functional Studies of DNA-Protein Interactions Using FRET Techniques. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_28
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