Abstract
Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h.
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References
FairmanWilliams, M.E., Guenther, U.P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010).
Singleton, M.R., Dillingham, M.S. & Wigley, D.B. Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50 (2007).
Jankowsky, A., Guenther, U.P. & Jankowsky, E. The RNA helicase database. Nucleic Acids Res. 39, D338–D341 (2011).
Jankowsky, E. RNA helicases at work: binding and rearranging. Trends Biochem. Sci. 36, 19–29 (2011).
Tani, H. et al. Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro. Biochem. Biophys. Res. Commun. 393, 131–136 (2010).
Ozes, A.R., Feoktistova, K., Avanzino, B.C. & Fraser, C.S. Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B. J. Mol. Biol. 412, 674–687 (2011).
Feoktistova, K., Tuvshintogs, E., Do, A. & Fraser, C.S. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc. Natl. Acad. Sci. USA 110, 13339–13344 (2013).
Brune, M., Hunter, J.L., Corrie, J.E. & Webb, M.R. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33, 8262–8271 (1994).
Brune, M. et al. Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37, 10370–10380 (1998).
Toseland, C.P. & Webb, M.R. Fluorescence tools to measure helicase activity in real time. Methods 51, 259–268 (2010).
Yodh, J.G., Schlierf, M. & Ha, T. Insight into helicase mechanism and function revealed through single-molecule approaches. Quart. Rev. Biophys. 43, 185–217 (2010).
Hilario, J. & Kowalczykowski, S.C. Visualizing protein-DNA interactions at the single-molecule level. Curr. Opin. Chem. Biol. 14, 15–22 (2010).
Fischer, C.J., Tomko, E.J., Wu, C.G. & Lohman, T.M. Fluorescence methods to study DNA translocation and unwinding kinetics by nucleic acid motors. Methods Mol. Biol. 875, 85–104 (2012).
Jankowsky, E. & Putnam, A. Duplex unwinding with DEAD-box proteins. Methods Mol. Biol. 587, 245–264 (2010).
Bjornson, K.P., Amaratunga, M., Moore, K.J. & Lohman, T.M. Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer. Biochemistry 33, 14306–14316 (1994).
Zhang, X.D., Dou, S.X., Xie, P., Wang, P.Y. & Xi, X.G. RecQ helicase-catalyzed DNA unwinding detected by fluorescence resonance energy transfer. Acta Biochim. Biophys. Sinica 37, 593–600 (2005).
Houston, P. & Kodadek, T. Spectrophotometric assay for enzyme-mediated unwinding of double-stranded DNA. Proc. Natl. Acad. Sci. USA 91, 5471–5474 (1994).
Belon, C.A. & Frick, D.N. Monitoring helicase activity with molecular beacons. BioTechniques 45, 433–440, 442 (2008).
Aydin, C., Mukherjee, S., Hanson, A.M., Frick, D.N. & Schiffer, C.A. The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities. Protein Sci. 22, 1786–1798 (2013).
Tyagi, S. & Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).
Hanson, A.M., Hernandez, J.J., Shadrick, W.R. & Frick, D.N. Identification and analysis of inhibitors targeting the hepatitis C virus NS3 helicase. Methods Enzymol. 511, 463–483 (2012).
Putnam, A. & Jankowsky, E. Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy. Methods Enzymol. 511, 1–27 (2012).
Moreira, B.G., You, Y., Behlke, M.A. & Owczarzy, R. Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Biochem. Biophys. Res. Commun. 327, 473–484 (2005).
Anderson, B.J., Larkin, C., Guja, K. & Schildbach, J.F. Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions. Methods Enzymol. 450, 253–272 (2008).
Liu, F., Putnam, A. & Jankowsky, E. ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding. Proc. Natl. Acad. Sci. USA 105, 20209–20214 (2008).
Rajagopal, V. & Lorsch, J.R. ATP and GTP hydrolysis assays (TLC). Methods Enzymol. 533, 325–334 (2013).
Young, C. & Karbstein, K. Analysis of cofactor effects on RNA helicases. Methods Enzymol. 511, 213–237 (2012).
Renosto, F., Seubert, P.A. & Segel, I.H. Adenosine 5′-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization. J. Biol. Chem. 259, 2113–2123 (1984).
Ingerman, E. & Nunnari, J. A continuous, regenerative coupled GTPase assay for dynamin-related proteins. Methods Enzymol. 404, 611–619 (2005).
Shutes, A. & Der, C.J. Real-time in vitro measurement of intrinsic and Ras GAP-mediated GTP hydrolysis. Methods Enzymol. 407, 9–22 (2006).
Pleiss, J.A., Derrick, M.L. & Uhlenbeck, O.C. T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates. RNA 4, 1313–1317 (1998).
Milligan, J.F., Groebe, D.R., Witherell, G.W. & Uhlenbeck, O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).
Vicens, Q., Gooding, A.R., Duarte, L.F. & Batey, R.T. Preparation of group I introns for biochemical studies and crystallization assays by native affinity purification. PLoS ONE 4, e6740 (2009).
Nilsen, T.W., Rio, D.C. & Ares, M. Jr. High-yield synthesis of RNA using T7 RNA polymerase and plasmid DNA or oligonucleotide templates. Cold Spring Harb. Protoc. 10.1101/pdb.prot078535 (2013).
Rogers, G.W. Jr., Richter, N.J., Lima, W.F. & Merrick, W.C. Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F. J. Biol. Chem. 276, 30914–30922 (2001).
Rio, D.C. Expression and purification of active recombinant T7 RNA polymerase from E. coli. Cold Spring Harb. Protoc. 10.1101/pdb.prot078527 (2013).
Green, M.R. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn. (Cold Spring Harbor Laboratory Press, 2012).
Sambrook, J., Maniatis, T. & Fritsch, E.F. Molecular Cloning: A Laboratory Manual 2nd edn. (Cold Spring Harbor Laboratory, 1989).
Acknowledgements
We thank B. Rad and S. Kowalczykowski and members of the Kowalczykowski laboratory for expert help and advice with developing this protocol. We also thank J. Hershey for his many insightful comments throughout the protocol development. We gratefully acknowledge M. Webb for his generous gift of PBP-MDCC and A. Do and E. Tuvshintogs for expert technical assistance. This work was supported by the US National Institutes of Health (NIH) through an NIHR01 grant (R01GM092927; to C.S.F.), an NIH training grant (T32 GM007377; to K.F.) and an American Heart Association and Myocarditis Foundation predoctoral fellowship (B.C.A.).
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A.R.O., K.F., B.C.A., E.P.B. and C.S.F. designed the study. A.R.O., K.F. and B.C.A. performed the experiments. A.R.O., K.F., B.C.A., E.P.B. and C.S.F. wrote the manuscript.
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Özeş, A., Feoktistova, K., Avanzino, B. et al. Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases. Nat Protoc 9, 1645–1661 (2014). https://doi.org/10.1038/nprot.2014.112
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DOI: https://doi.org/10.1038/nprot.2014.112
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