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Single Molecule Analysis pp 217–239Cite as

Single Molecule FRET Analysis of DNA Binding Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1665))

Abstract

The complex binding dynamics between DNA and proteins are often obscured by ensemble averaging effects in conventional biochemical experiments. Single-molecule fluorescence methods are powerful tools to investigate DNA–protein interaction dynamics in real time. In this chapter, we focus on using single-molecule Förster Resonance Energy Transfer (smFRET) to probe the binding dynamics of individual proteins on single DNA molecules. We provide a detailed discussion of total internal reflection fluorescence (TIRF) instrument design, nucleic acid labeling with fluorophores, flow cell surface passivation, and data analysis methods.

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References

  1. Wuite GJ, Smith SB, Young M, Keller D, Bustamante C (2000) Single-molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 404(6773):103–106. doi:10.1038/35003614

    Article  CAS  PubMed  Google Scholar 

  2. Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang JX, Le Grice SF, Zhuang X (2008) Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature 453(7192):184–189. doi:10.1038/nature06941

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kim S, Schroeder CM, Xie XS (2010) Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates. J Mol Biol 395(5):995–1006. doi:10.1016/j.jmb.2009.11.072

    Article  CAS  PubMed  Google Scholar 

  4. Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM (2005) Direct observation of base-pair stepping by RNA polymerase. Nature 438(7067):460–465. doi:10.1038/nature04268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kapanidis AN, Margeat E, Ho SO, Kortkhonjia E, Weiss S, Ebright RH (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314(5802):1144–1147. doi:10.1126/science.1131399

    Article  PubMed  PubMed Central  Google Scholar 

  6. Herbert KM, Greenleaf WJ, Block SM (2008) Single-molecule studies of RNA polymerase: motoring along. Annu Rev Biochem 77:149–176. doi:10.1146/annurev.biochem.77.073106.100741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sorokina M, Koh HR, Patel SS, Ha T (2009) Fluorescent lifetime trajectories of a single fluorophore reveal reaction intermediates during transcription initiation. J Am Chem Soc 131(28):9630–9631. doi:10.1021/ja902861f

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lee KS, Balci H, Jia H, Lohman TM, Ha T (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1878. doi:10.1038/ncomms2882

    Article  PubMed  PubMed Central  Google Scholar 

  9. Byrd AK, Matlock DL, Bagchi D, Aarattuthodiyil S, Harrison D, Croquette V, Raney KD (2012) Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase. J Mol Biol 420(3):141–154. doi:10.1016/j.jmb.2012.04.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cheng W, Arunajadai SG, Moffitt JR, Tinoco I Jr, Bustamante C (2011) Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase. Science 333(6050):1746–1749. doi:10.1126/science.1206023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ha T, Rasnik I, Cheng W, Babcock HP, Gauss GH, Lohman TM, Chu S (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419(6907):638–641. doi:10.1038/nature01083

    Article  CAS  PubMed  Google Scholar 

  12. Chaurasiya KR, Ruslie C, Silva MC, Voortman L, Nevin P, Lone S, Beuning PJ, Williams MC (2013) Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III alpha binding to ssDNA. Nucleic Acids Res 41(19):8959–8968. doi:10.1093/nar/gkt648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chaurasiya KR, McCauley MJ, Wang W, Qualley DF, Wu T, Kitamura S, Geertsema H, Chan DS, Hertz A, Iwatani Y, Levin JG, Musier-Forsyth K, Rouzina I, Williams MC (2014) Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein. Nat Chem 6(1):28–33. doi:10.1038/nchem.1795

    Article  CAS  PubMed  Google Scholar 

  14. Driessen RP, Sitters G, Laurens N, Moolenaar GF, Wuite GJ, Goosen N, Dame RT (2014) Effect of temperature on the intrinsic flexibility of DNA and its interaction with architectural proteins. Biochemistry 53(41):6430–6438. doi:10.1021/bi500344j

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Senavirathne G, Bertram JG, Jaszczur M, Chaurasiya KR, Pham P, Mak CH, Goodman MF, Rueda D (2015) Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution. Nat Commun 6:10209. doi:10.1038/ncomms10209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Brouwer I, Sitters G, Candelli A, Heerema SJ, Heller I, de Melo AJ, Zhang H, Normanno D, Modesti M, Peterman EJ, Wuite GJ (2016) Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA. Nature 535(7613):566–569. doi:10.1038/nature18643

    Article  CAS  PubMed  Google Scholar 

  17. Funatsu T, Harada Y, Tokunaga M, Saito K, Yanagida T (1995) Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374(6522):555–559. doi:10.1038/374555a0

    Article  CAS  PubMed  Google Scholar 

  18. Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065. doi:10.1126/science.1084398

    Article  CAS  PubMed  Google Scholar 

  19. Verbrugge S, Lansky Z, Peterman EJ (2009) Kinesin’s step dissected with single-motor FRET. Proc Natl Acad Sci U S A 106(42):17741–17746. doi:10.1073/pnas.0905177106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Nguyen DC, Keller RA, Jett JH, Martin JC (1987) Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser-induced fluorescence. Anal Chem 59(17):2158–2161. doi:10.1021/ac00144a032

    Article  CAS  PubMed  Google Scholar 

  21. Hirschfeld T (1976) Optical microscopic observation of single small molecules. Appl Opt 15(12):2965–2966. doi:10.1364/ao.15.002965

    Article  CAS  PubMed  Google Scholar 

  22. Förster T (1948) Intermolecular energy migration and fluorescence. Ann Phys 2:55–75

    Article  Google Scholar 

  23. Clegg RM (1992) Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol 211:353–388

    Article  CAS  PubMed  Google Scholar 

  24. Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7(9):730–734. doi:10.1038/78948

    Article  CAS  PubMed  Google Scholar 

  25. Sofia SJ, Premnath VV, Merrill EW (1998) Poly(ethylene oxide) grafted to silicon surfaces: grafting density and protein adsorption. Macromolecules 31(15):5059–5070. doi:10.1021/ma971016l

    Article  CAS  PubMed  Google Scholar 

  26. Jeyachandran YL, Mielczarski JA, Mielczarski E, Rai B (2010) Efficiency of blocking of non-specific interaction of different proteins by BSA adsorbed on hydrophobic and hydrophilic surfaces. J Colloid Interface Sci 341(1):136–142. doi:10.1016/j.jcis.2009.09.007

    Article  CAS  PubMed  Google Scholar 

  27. Hua B, Han KY, Zhou R, Kim H, Shi X, Abeysirigunawardena SC, Jain A, Singh D, Aggarwal V, Woodson SA, Ha T (2014) An improved surface passivation method for single-molecule studies. Nat Methods 11(12):1233–1236. doi:10.1038/nmeth.3143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank all the members of the David Rueda laboratory for training in single-molecule FRET methods. This work was supported in part by the Human Frontiers Science Program [RGP0014/2014] and the Netherlands Organization for Scientific Research [VICI 016.160.613].

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Authors and Affiliations

  1. Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333CC, Leiden, The Netherlands

    Kathy R. Chaurasiya & Remus T. Dame

Authors
  1. Kathy R. Chaurasiya
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  2. Remus T. Dame
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Corresponding author

Correspondence to Kathy R. Chaurasiya .

Editor information

Editors and Affiliations

  1. Department of Physics & Astronomy, Vrije Universiteit, Amsterdam, The Netherlands

    Erwin J. G. Peterman

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Chaurasiya, K.R., Dame, R.T. (2018). Single Molecule FRET Analysis of DNA Binding Proteins. In: Peterman, E. (eds) Single Molecule Analysis. Methods in Molecular Biology, vol 1665. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7271-5_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7271-5_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7270-8

  • Online ISBN: 978-1-4939-7271-5

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