Insight into helicase mechanism and function revealed through single-molecule approaches | Quarterly Reviews of Biophysics | Cambridge Core (original) (raw)

Abstract

Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

References

Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R. & Block, S. M. (2005). Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465.Google Scholar

Ali, J. A. & Lohman, T. M. (1997). Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275, 377–380.Google Scholar

Ali, J. A., Maluf, N. K. & Lohman, T. M. (1999). An oligomeric form of E. coli UvrD is required for optimal helicase activity. Journal of Molecular Biology 293, 815–834.CrossRefGoogle ScholarPubMed

Aregger, R. & Klostermeier, D. (2009). The DEAD box helicase YxiN maintains a closed conformation during ATP hydrolysis. Biochemistry 48, 10679–10681.Google Scholar

Arumugam, S. R., Lee, T. H. & Benkovic, S. J. (2009). Investigation of stoichiometry of T4 bacteriophage helicase loader protein (gp59). Journal of Biological Chemistry 284, 29283–29289.Google Scholar

Berger, J. M. (2008). SnapShot: nucleic acid helicases and translocases. Cell 134, 888.Google Scholar

Bertram, R. D., Hayes, C. J. & Soultanas, P. (2002). Vinylphosphonate internucleotide linkages inhibit the activity of PcrA DNA helicase. Biochemistry 41, 7725–7731.Google Scholar

Betterton, M. D. & Julicher, F. (2003). A motor that makes its own track: helicase unwinding of DNA. Physical Review Letters 91, 258103.CrossRefGoogle ScholarPubMed

Betterton, M. D. & Julicher, F. (2005). Opening of nucleic-acid double strands by helicases: active versus passive opening. Physical Review E, Statistical, Nonlinear, and Soft Matter Physics 71, 011904.Google Scholar

Bianco, P. R., Brewer, L. R., Corzett, M., Balhorn, R., Yeh, Y., Kowalczykowski, S. C. & Baskin, R. J. (2001). Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378.Google Scholar

Brendza, K. M., Cheng, W., Fischer, C. J., Chesnik, M. A., Niedziela-Majka, A. & Lohman, T. M. (2005). Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain. Proceedings of the National Academy of Sciences USA 102, 10076–10081.CrossRefGoogle ScholarPubMed

Brosh, R. M. Jr., Li, J. L., Kenny, M. K., Karow, J. K., Cooper, M. P., Kureekattil, R. P., Hickson, I. D. & Bohr, V. A. (2000). Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity. Journal of Biological Chemistry 275, 23500–23508.CrossRefGoogle ScholarPubMed

Chen, Y., Potratz, J. P., Tijerina, P., Del Campo, M., Lambowitz, A. M. & Russell, R. (2008). DEAD-box proteins can completely separate an RNA duplex using a single ATP. Proceedings of the National Academy of Sciences USA 105, 20203–20208.CrossRefGoogle ScholarPubMed

Cheng, W., Brendza, K. M., Gauss, G. H., Korolev, S., Waksman, G. & Lohman, T. M. (2002). The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity. Proceedings of the National Academy of Sciences USA 99, 16006–16011.Google Scholar

Cheng, W., Dumont, S., Tinoco, I. Jr. & Bustamante, C. (2007). NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork. Proceedings of the National Academy of Sciences USA 104, 13954–13959.CrossRefGoogle ScholarPubMed

Delagoutte, E. & von Hippel, P. H. (2001). Molecular mechanisms of the functional coupling of the helicase (gp41) and polymerase (gp43) of bacteriophage T4 within the DNA replication fork. Biochemistry 40, 4459–4477.CrossRefGoogle ScholarPubMed

Dessinges, M. N., Lionnet, T., Xi, X. G., Bensimon, D. & Croquette, V. (2004). Single-molecule assay reveals strand switching and enhanced processivity of UvrD. Proceedings of the National Academy of Sciences USA 101, 6439–6444.CrossRefGoogle ScholarPubMed

Dillingham, M. S., Wigley, D. B. & Webb, M. R. (2000). Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed. Biochemistry 39, 205–212.CrossRefGoogle ScholarPubMed

Dillingham, M. S., Wigley, D. B. & Webb, M. R. (2002). Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine. Biochemistry 41, 643–651.Google Scholar

Dohoney, K. M. & Gelles, J. (2001). Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules. Nature 409, 370–374.CrossRefGoogle ScholarPubMed

Dong, F., Weitzel, S. E. & von Hippel, P. H. (1996). A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis. Proceedings of the National Academy of Sciences USA 93, 14456–14461.CrossRefGoogle ScholarPubMed

Dumont, S., Cheng, W., Serebrov, V., Beran, R. K., Tinoco, I. Jr., Pyle, A. M. & Bustamante, C. (2006). RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108.Google Scholar

Enemark, E. J. & Joshua-Tor, L. (2008). On helicases and other motor proteins. Current Opinion in Structural Biology 18, 243–257.Google Scholar

Essevaz-Roulet, B., Bockelmann, U. & Heslot, F. (1997). Mechanical separation of the complementary strands of DNA. Proceedings of the National Academy of Sciences USA 94, 11935–11940.Google Scholar

Fan, H. F. & Li, H. W. (2009). Studying RecBCD helicase translocation along Chi-DNA using tethered particle motion with a stretching force. Biophysical Journal 96, 1875–1883.Google Scholar

Fischer, C. J. & Lohman, T. M. (2004). ATP-dependent translocation of proteins along single-stranded DNA: models and methods of analysis of pre-steady state kinetics. Journal of Molecular Biology 344, 1265–1286.CrossRefGoogle ScholarPubMed

Fischer, C. J., Maluf, N. K. & Lohman, T. M. (2004). Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA. Journal of Molecular Biology 344, 1287–1309.CrossRefGoogle ScholarPubMed

Ha, T., Rasnik, I., Cheng, W., Babcock, H. P., Gauss, G. H., Lohman, T. M. & Chu, S. (2002). Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641.CrossRefGoogle ScholarPubMed

Hamdan, S. M., Johnson, D. E., Tanner, N. A., Lee, J. B., Qimron, U., Tabor, S., van Oijen, A. M. & Richardson, C. C. (2007). Dynamic DNA helicase–DNA polymerase interactions assure processive replication fork movement. Molecular Cell 27, 539–549.CrossRefGoogle ScholarPubMed

Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C. & van Oijen, A. M. (2009). Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336–339.Google Scholar

Handa, N., Bianco, P. R., Baskin, R. J. & Kowalczykowski, S. C. (2005). Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition. Molecular Cell 17, 745–750.Google Scholar

Henn, A., Medalia, O., Shi, S. P., Steinberg, M., Franceschi, F. & Sagi, I. (2001). Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy. Proceedings of the National Academy of Sciences USA 98, 5007–5012.CrossRefGoogle ScholarPubMed

Hilbert, M., Karow, A. R. & Klostermeier, D. (2009). The mechanism of ATP-dependent RNA unwinding by DEAD box proteins. Biological Chemistry 390, 1237–1250.CrossRefGoogle ScholarPubMed

Honda, M., Park, J., Pugh, R. A., Ha, T. & Spies, M. (2009). Single-molecule analysis reveals differential effect of ssDNA-binding proteins on DNA translocation by XPD helicase. Molecular Cell 35, 694–703.CrossRefGoogle ScholarPubMed

Hopfner, K. P. & Michaelis, J. (2007). Mechanisms of nucleic acid translocases: lessons from structural biology and single-molecule biophysics. Current Opinion in Structural Biology 17, 87–95.CrossRefGoogle ScholarPubMed

Jankowsky, E., Gross, C. H., Shuman, S. & Pyle, A. M. (2000). The DExH protein NPH-II is a processive and directional motor for unwinding RNA. Nature 403, 447–451.CrossRefGoogle ScholarPubMed

Janscak, P., Garcia, P. L., Hamburger, F., Makuta, Y., Shiraishi, K., Imai, Y., Ikeda, H. & Bickle, T. A. (2003). Characterization and mutational analysis of the RecQ core of the bloom syndrome protein. Journal of Molecular Biology 330, 29–42.CrossRefGoogle ScholarPubMed

Johnson, D. S., Bai, L., Smith, B. Y., Patel, S. S. & Wang, M. D. (2007). Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell 129, 1299–1309.Google Scholar

Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. (2008). Advances in single-molecule fluorescence methods for molecular biology. Annual Review of Biochemistry 77, 51–76.Google Scholar

Karow, A. R. & Klostermeier, D. (2009). A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN. Nucleic Acids Research 37, 4464–4471.Google Scholar

Lee, J. B., Hite, R. K., Hamdan, S. M., Xie, X. S., Richardson, C. C. & van Oijen, A. M. (2006). DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624.Google Scholar

Lee, J. Y. & Yang, W. (2006). UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360.CrossRefGoogle Scholar

Linden, M. H., Hartmann, R. K. & Klostermeier, D. (2008). The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity. Nucleic Acids Research 36, 5800–5811.Google Scholar

Lionnet, T., Dawid, A., Bigot, S., Barre, F. X., Saleh, O. A., Heslot, F., Allemand, J. F., Bensimon, D. & Croquette, V. (2006). DNA mechanics as a tool to probe helicase and translocase activity. Nucleic Acids Research 34, 4232–4244.Google Scholar

Lionnet, T., Spiering, M. M., Benkovic, S. J., Bensimon, D. & Croquette, V. (2007). Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism. Proceedings of the National Academy of Sciences USA 104, 19790–19795.Google Scholar

Liu, F., Putnam, A. & Jankowsky, E. (2008). ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding. Proceedings of the National Academy of Sciences USA 105, 20209–20214.Google Scholar

Lohman, T. M. & Bjornson, K. P. (1996). Mechanisms of helicase-catalyzed DNA unwinding. Annual Review of Biochemistry 65, 169–214.CrossRefGoogle ScholarPubMed

Lohman, T. M., Tomko, E. J. & Wu, C. G. (2008). Non-hexameric DNA helicases and translocases: mechanisms and regulation. Nature Reviews Molecular Cell Biology 9, 391–401.Google Scholar

Lucius, A. L., Maluf, N. K., Fischer, C. J. & Lohman, T. M. (2003). General methods for analysis of sequential “n-step” kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA _unwindin_g. Biophysical Journal 85, 2224–2239.Google Scholar

Lucius, A. L., Vindigni, A., Gregorian, R., Ali, J. A., Taylor, A. F., Smith, G. R. & Lohman, T. M. (2002). DNA unwinding step size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies. Journal of Molecular Biology 324, 409–428.Google Scholar

Maluf, N. K., Fischer, C. J. & Lohman, T. M. (2003). A dimer of Escherichia coli UvrD is the active form of the helicase in vitro. J. Mol. Biol. 325, 913–935.Google Scholar

Manosas, M., Spiering, M. M., Zhuang, Z., Benkovic, S. J. & Croquette, V. (2009). Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome. Nature Chemical Biology 5, 904–912.Google Scholar

Marsden, S., Nardelli, M., Linder, P. & McCarthy, J. E. (2006). Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation. Journal of Molecular Biology 361, 327–35.Google Scholar

Moffitt, J. R., Chemla, Y. R., Aathavan, K., Grimes, S., Jardine, P. J., Anderson, D. L. & Bustamante, C. (2009). Intersubunit coordination in a homomeric ring ATPase. Nature 457, 446–450.CrossRefGoogle Scholar

Myong, S., Bruno, M. M., Pyle, A. M. & Ha, T. (2007). Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317, 513–516.Google Scholar

Myong, S., Cui, S., Cornish, P. V., Kirchhofer, A., Gack, M. U., Jung, J. U., Hopfner, K. P. & Ha, T. (2009). Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on double-stranded RNA. Science 323, 1070–1074.CrossRefGoogle Scholar

Myong, S., Rasnik, I., Joo, C., Lohman, T. M. & Ha, T. (2005). Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325.Google Scholar

Niedziela-Majka, A., Chesnik, M. A., Tomko, E. J. & Lohman, T. M. (2007). Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro. Journal of Biological Chemistry 282, 27076–27085.Google Scholar

Pandey, M., Syed, S., Donmez, I., Patel, G., Ha, T. & Patel, S. S. (2009). Coordinating DNA replication by means of priming loop and differential synthesis rate. Nature 462, 940–943.CrossRefGoogle ScholarPubMed

Perkins, T. T., Li, H. W., Dalal, R. V., Gelles, J. & Block, S. M. (2004). Forward and reverse motion of single RecBCD molecules on DNA. Biophysical Journal 86, 1640–1648.CrossRefGoogle ScholarPubMed

Phillips, R., Kondev, J. & Theriot, J. (2008). Physical Biology of the Cell. New York, NY: Garland Science, Taylor & Francis Group.Google Scholar

Pugh, R. A., Lin, Y., Eller, C., Leesley, H., Cann, I. K. & Spies, M. (2008). Ferroplasma acidarmanus RPA2 facilitates efficient unwinding of forked DNA substrates by monomers of FacXPD helicase. Journal of Molecular Biology 383, 982–998.CrossRefGoogle ScholarPubMed

Pyle, A. M. (2008). Translocation and unwinding mechanisms of RNA and DNA helicases. Annual Review of Biophysics 37, 317–336.Google Scholar

Ramanathan, S. P., van Aelst, K., Sears, A., Peakman, L. J., Diffin, F. M., Szczelkun, M. D. & Seidel, R. (2009). Type III restriction enzymes communicate in 1D without looping between their target sites. Proceedings of the National Academy of Sciences USA 106, 1748–1753.Google Scholar

Rasnik, I., Myong, S. & Ha, T. (2006). Unraveling helicase mechanisms one molecule at a time. Nucleic Acids Research 34, 4225–4231.CrossRefGoogle ScholarPubMed

Ratcliff, G. C. & Erie, D. A. (2001). A novel single-molecule study to determine protein–protein association constants. Journal of the American Chemical Society 123, 5632–5635.CrossRefGoogle ScholarPubMed

Rothenberg, E., Trakselis, M. A., Bell, S. D. & Ha, T. (2007). MCM forked substrate specificity involves dynamic interaction with the 5′-tail. Journal of Biological Chemistry 282, 34229–34234.CrossRefGoogle Scholar

Roy, R., Hohng, S. & Ha, T. (2008). A practical guide to single-molecule FRET. Nature Methods 5, 507–516.Google Scholar

Schrock, R. D. & Alberts, B. (1996). Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork. Journal of Biological Chemistry 271, 16678–16682.Google Scholar

Serebrov, V., Beran, R. K. & Pyle, A. M. (2009). Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase. Journal of Biological Chemistry 284, 2512–2521.CrossRefGoogle ScholarPubMed

Serebrov, V. & Pyle, A. M. (2004). Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430, 476–480.CrossRefGoogle ScholarPubMed

Singleton, M. R., Dillingham, M. S. & Wigley, D. B. (2007). Structure and mechanism of helicases and nucleic acid translocases. Annual Review of Biochemistry 76, 23–50.CrossRefGoogle ScholarPubMed

Singleton, M. R. & Wigley, D. B. (2002). Modularity and specialization in superfamily 1 and 2 helicases. Journal of Bacteriology 184, 1819–1826.CrossRefGoogle ScholarPubMed

Slatter, A. F., Thomas, C. D. & Webb, M. R. (2009). PcrA helicase tightly couples ATP hydrolysis to unwinding double-stranded DNA, modulated by the initiator protein for plasmid replication, RepD. Biochemistry 48, 6326–6334.Google Scholar

Spies, M., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. (2007). RecBCD enzyme switches lead motor subunits in response to chi recognition. Cell 131, 694–705.Google Scholar

Spies, M., Bianco, P. R., Dillingham, M. S., Handa, N., Baskin, R. J. & Kowalczykowski, S. C. (2003). A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase. Cell 114, 647–654.Google Scholar

Stano, N. M., Jeong, Y. J., Donmez, I., Tummalapalli, P., Levin, M. K. & Patel, S. S. (2005). DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435, 370–373.Google Scholar

Sun, B., Wei, K. J., Zhang, B., Zhang, X. H., Dou, S. X., Li, M. & Xi, X. G. (2008). Impediment of E. coli UvrD by DNA-destabilizing force reveals a strained-inchworm mechanism of DNA unwinding. EMBO Journal 27, 3279–3287.CrossRefGoogle ScholarPubMed

Tanner, N. A., Hamdan, S. M., Jergic, S., Loscha, K. V., Schaeffer, P. M., Dixon, N. E. & van Oijen, A. M. (2008). Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nature Structural and Molecular Biology 15, 170–176.Google Scholar

Tanner, N. A., Loparo, J. J., Hamdan, S. M., Jergic, S., Dixon, N. E. & van Oijen, A. M. (2009). Real-time single-molecule observation of rolling-circle DNA replication. Nucleic Acids Research 37, e27.Google Scholar

Theissen, B., Karow, A. R., Kohler, J., Gubaev, A. & Klostermeier, D. (2008). Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase. Proceedings of the National Academy of Sciences USA 105, 548–553.Google Scholar

Tomko, E. J., Fischer, C. J., Niedziela-Majka, A. & Lohman, T. M. (2007). A nonuniform stepping mechanism for E. coli UvrD monomer translocation along single-stranded DNA. Molecular Cell 26, 335–347.Google Scholar

Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. (1999). Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84.CrossRefGoogle ScholarPubMed

Wickersham, C. E., Cash, K. J., Pfeil, S. H., Bruck, I., Kaplan, D. L., Plaxco, K. W. & Lipman, E. A. (2010). Tracking a molecular motor with a nanoscale optical encoder. NanoLetters, 10(3), 1022–1027.Google Scholar

Xi, J., Zhang, Z., Zhuang, Z., Yang, J., Spiering, M. M., Hammes, G. G. & Benkovic, S. J. (2005a). Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59–gp43–DNA complex to initiate assembly of a fully functional replisome. Biochemistry 44, 7747–7756.CrossRefGoogle ScholarPubMed

Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G. & Benkovic, S. J. (2005b). Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly. Biochemistry 44, 2305–2318.Google Scholar

Yang, Y., Dou, S. X., Ren, H., Wang, P. Y., Zhang, X. D., Qian, M., Pan, B. Y. & Xi, X. G. (2008). Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding. Nucleic Acids Research 36, 1976–1989.Google Scholar

Yodh, J. G., Stevens, B. C., Kanagaraj, R., Janscak, P. & Ha, T. (2009). BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation. EMBO Journal 28, 405–416.CrossRefGoogle ScholarPubMed

Zhang, W., Dillingham, M. S., Thomas, C. D., Allen, S., Roberts, C. J. & Soultanas, P. (2007). Directional loading and stimulation of PcrA helicase by the replication initiator protein RepD. Journal of Molecular Biology 371, 336–348.Google Scholar

Zhang, Z., Spiering, M. M., Trakselis, M. A., Ishmael, F. T., Xi, J., Benkovic, S. J. & Hammes, G. G. (2005). Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies. Proceedings of the National Academy of Sciences USA 102, 3254–3259.CrossRefGoogle ScholarPubMed