Abstract
Helicases are nucleic acid-dependent ATPases which can bind and remodel nucleic acids, protein–nucleic acid complexes, or both. They are involved in almost every step in cells related to nucleic acid metabolisms, including DNA replication and repair, transcription, RNA maturation and splicing, and nuclear export processes. Using single-molecule fluorescence-force spectroscopy, we have previously directly observed helicase translocation on long single-stranded DNA and revealed that two monomers of UvrD helicase are required for the initiation of unwinding function. Here, we present the details of fluorescence-force spectroscopy instrumentation, calibration, and activity assays in detail for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein–nucleic acid interaction. These single-molecule approaches are generally applicable to many other protein–nucleic acid systems.
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References
Lu HP, Xun L, Xie XS (1998) Single-molecule enzymatic dynamics. Science 282(5395):1877–1882. doi:10.1126/science.282.5395.1877
Robison AD, Finkelstein IJ (2014) High-throughput single-molecule studies of protein–DNA interactions. FEBS Lett 588(19):3539–3546. doi:10.1016/j.febslet.2014.05.021
Ilya JF, Eric CG (2013) Molecular traffic jams on DNA. Annu Rev Biophys 42(1):241–263. doi:10.1146/annurev-biophys-083012-130304
Ha T, Enderle T, Ogletree DF et al (1996) Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci U S A 93(13):6264–6268
Roy R, Hohng S, Ha T (2008) A practical guide to single molecule FRET. Nat Methods 5(6):507–516. doi:10.1038/nmeth.1208
Visscher K, Gross SP, Block SM (1996) Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J Sel Top Quantum Electron 2(4):1066–1076. doi:10.1109/2944.577338
Comstock MJ, Whitley KD, Jia H et al (2015) Direct observation of structure-function relationship in a nucleic acid–processing enzyme. Science 348(6232):352–354. doi:10.1126/science.aaa0130
Moffitt JR, Chemla YR, Izhaky D et al (2006) Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc Natl Acad Sci U S A 103(24):9006–9011. doi:10.1073/pnas.0603342103
Woodside MT, Anthony PC, Behnke-Parks WM et al (2006) Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science 314(5801):1001–1004. doi:10.1126/science.1133601
Weiss S (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat Struct Mol Biol 7(9):724–729
Greenleaf WJ, Woodside MT, Block SM (2007) High-resolution, single-molecule measurements of biomolecular motion. Annu Rev Biophys Biomol Struct 36:171
Joo C, Balci H, Ishitsuka Y et al (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51
Walter NG, Huang CY, Manzo AJ et al (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475
Matson SW, Bean DW, George JW (1994) DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. Bioessays 16(1):13–22. doi:10.1002/bies.950160103
Schmid SR, Linder P (1992) D-E-A-D protein family of putative RNA helicases. Mol Microbiol 6(3):283–292. doi:10.1111/j.1365-2958.1992.tb01470.x
Jankowsky E, Gross CH, Shuman S et al (2001) Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291(5501):121–125. doi:10.1126/science.291.5501.121
Lohman TM, Bjornson KP (1996) Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem 65(1):169–214. doi:10.1146/annurev.bi.65.070196.001125
Tuteja N, Tuteja R (2004) Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery. Eur J Biochem 271(10):1835–1848. doi:10.1111/j.1432-1033.2004.04093.x
Singleton MR, Dillingham MS, Wigley DB (2007) Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 76:23–50
Mackintosh SG, Raney KD (2006) DNA unwinding and protein displacement by superfamily 1 and superfamily 2 helicases. Nucleic Acids Res 34(15):4154–4159. doi:10.1093/nar/gkl501
Donmez I, Patel SS (2006) Mechanisms of a ring shaped helicase. Nucleic Acids Res 34(15):4216–4224. doi:10.1093/nar/gkl508
Jankowsky E, Fairman ME (2007) RNA helicases—one fold for many functions. Curr Opin Struct Biol 17(3):316–324. doi:10.1016/j.sbi.2007.05.007
Vindigni A (2007) Biochemical, biophysical, and proteomic approaches to study DNA helicases. Mol Biosyst 3(4):266–274. doi:10.1039/B616145F
Lee KS, Balci H, Jia H et al (2013) Direct imaging of single UvrD helicase dynamics on long single-stranded DNA. Nat Commun 4:1878. doi:10.1038/ncomms2882
Lin C-T, Tritschler F, Suk Lee K et al (2014) Single-molecule imaging reveals the translocation dynamics of hepatitis C virus NS3 helicase. Biophys J 106(2):72a. doi:10.1016/j.bpj.2013.11.474
Lee KS (2013) Fluorescence imaging of single molecule dynamics on long single stranded DNA. (Doctor of Philosophy Doctoral dissertation), University of Illinois at Urbana-Champaign. http://hdl.handle.net/2142/42452
Lee KS, Marciel AB, Kozlov AG et al (2014) Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. J Mol Biol 426(13):2413–2421. doi:10.1016/j.jmb.2014.04.023
Brockman C, Kim SJ, Schroeder CM (2011) Direct observation of single flexible polymers using single stranded DNA. Soft Matter 7(18):8005–8012. doi:10.1039/C1SM05297G
Zhou R, Schlierf M, Ha T (2010) Chapter Sixteen - Force–fluorescence spectroscopy at the single-molecule level. In: Nils GW (ed) Methods in enzymology, vol 475. Academic, New York, NY, pp 405–426. doi:10.1016/S0076-6879(10)75016-3
Selvin PR, Ha T (2008) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Hua B, Han KY, Zhou R et al (2014) An improved surface passivation method for single-molecule studies. Nat Methods 11(12):1233–1236. doi:10.1038/nmeth.3143, http://www.nature.com/nmeth/journal/v11/n12/abs/nmeth.3143.html - supplementary-information
Rice SE, Purcell TJ, Spudich JA (2003) [6] Building and using optical traps to study properties of molecular motors. In: Nils GW (ed) Methods in enzymology, vol 361. Academic, New York, NY, pp 112–133. doi:10.1016/S0076-6879(03)61008-6
Berg-Sørensen K, Flyvbjerg H (2004) Power spectrum analysis for optical tweezers. Rev Sci Instrum 75(3):594–612. doi:10.1063/1.1645654
Berg-Sørensen K, Peterman EJG, Weber T et al (2006) Power spectrum analysis for optical tweezers. II: laser wavelength dependence of parasitic filtering, and how to achieve high bandwidth. Rev Sci Instrum 77(6):063106. doi:10.1063/1.2204589
Kubo R, Toda M, Hashitsume N (1991) Statistical physics II: nonequilibrium statistical mechanics. Springer, Berlin
Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75(9):2787–2809. doi:10.1063/1.1785844
Toprak E, Balci H, Blehm BH et al (2007) Three-dimensional particle tracking via bifocal imaging. Nano Lett 7(7):2043–2045. doi:10.1021/nl0709120
Baumann CG, Smith SB, Bloomfield VA et al (1997) Ionic effects on the elasticity of single DNA molecules. Proc Natl Acad Sci U S A 94(12):6185–6190
Yildiz A, Forkey JN, McKinney SA et al (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
Swoboda M, Henig J, Cheng H-M et al (2012) Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments. ACS Nano 6(7):6364–6369. doi:10.1021/nn301895c
van Dijk MA, Kapitein LC, van Mameren J et al (2004) Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. J Phys Chem B 108(20):6479–6484. doi:10.1021/jp049805+
Lang MJ, Asbury CL, Shaevitz JW et al (2002) An automated two-dimensional optical force clamp for single molecule studies. Biophys J 83(1):491–501. doi:10.1016/S0006-3495(02)75185-0
Köhler A (1984) New method of illumination for phomicrographical purposes. J R Microsc Soc 14:261–262
Acknowledgements
T. H. is an Investigator of the Howard Hughes Medical Institute. This work is supported by NIH grant GM065367 and NSF grants PHY-1430124 to T. H. We would like to thank Olivia Yang for proofreading the manuscript and Dr. Kyung Suk Lee for constructing the original instrument and training.
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Lin, CT., Ha, T. (2017). Probing Single Helicase Dynamics on Long Nucleic Acids Through Fluorescence-Force Measurement. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 1486. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6421-5_11
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DOI: https://doi.org/10.1007/978-1-4939-6421-5_11
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