In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA1,2,3,4. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as EcoRIE111Q, lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein–protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.
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Jankowsky, E., Gross, C., Shuman, S. & Pyle, A. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125 (2001)
Marquis, K. A. et al. SpoIIIE strips proteins off the DNA during chromosome translocation. Genes Dev. 22, 1786–1795 (2008)
Krejci, L. et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423, 305–309 (2003)
Guy, C. P. et al. Rep provides a second motor at the replisome to promote duplication of protein-bound DNA. Mol. Cell 36, 654–666 (2009)
Singleton, M. R., Dillingham, M., Gaudier, M., Kowalczykowski, S. & Wigley, D. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004)
Bianco, P. R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001)
Spies, M., Amitani, I., Baskin, R. & Kowalczykowski, S. RecBCD enzyme switches lead motor subunits in response to chi recognition. Cell 131, 694–705 (2007)
Taylor, A. F. & Smith, G. R. RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity. Nature 423, 889–893 (2003)
Visnapuu, M.-L. & Greene, E. Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition. Nature Struct. Mol. Biol. 16, 1056–1062 (2009)
Ishihama, A. Functional modulation of Escherichia coli RNA polymerase. Annu. Rev. Microbiol. 54, 499–518 (2000)
Herbert, K. M., Greenleaf, W. J. & Block, S. M. Single-molecule studies of RNA polymerase: motoring along. Annu. Rev. Biochem. 77, 149–176 (2008)
Liu, B., Wong, M. & Alberts, B. A transcribing RNA polymerase molecule survives DNA replication without aborting its growing RNA chain. Proc. Natl Acad. Sci. USA 91, 10660–10664 (1994)
Liu, B., Wong, M., Tinker, R., Geiduschek, E. & Alberts, B. The DNA replication fork can pass RNA polymerase without displacing the nascent transcript. Nature 366, 33–39 (1993)
Liu, B. & Alberts, B. Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex. Science 267, 1131–1137 (1995)
Pomerantz, R. T. & O’Donnell, M. The replisome uses mRNA as a primer after colliding with RNA polymerase. Nature 456, 762–766 (2008)
Pomerantz, R. T. & O’Donnell, M. Direct restart of a replication fork stalled by a head-on RNA polymerase. Science 327, 590–592 (2010)
Wright, D. J., King, K. & Modrich, P. The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease. J. Biol. Chem. 264, 11816–11821 (1989)
Epshtein, V. & Toulm, È. F. Rahmouni, A. Borukhov, S. & Nudler, E. Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 22, 4719–4727 (2003)
Nudler, E., Kashlev, M., Nikiforov, V. & Goldfarb, A. Coupling between transcription termination and RNA polymerase inchworming. Cell 81, 351–357 (1995)
Pavco, P. A. & Steege, D. A. Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein. Nucleic Acids Res. 19, 4639–4646 (1991)
Byrd, A. K. & Raney, K. D. Displacement of a DNA binding protein by Dda helicase. Nucleic Acids Res. 34, 3020–3029 (2006)
Noom, M. C., van den Broek, B., van Mameren, J. & Wuite, G. J. L. Visualizing single DNA-bound proteins using DNA as a scanning probe. Nature Methods 4, 1031–1036 (2007)
Sadler, J. R., Sasmor, H. & Betz, J. L. A perfectly symmetric lac operator binds the lac repressor very tightly. Proc. Natl Acad. Sci. USA 80, 6785–6789 (1983)
Lin, S.-Y. & Riggs, A. D. Lac repressor binding to DNA not containing the lac operator and to synthetic poly dAT. Nature 228, 1184–1186 (1970)
Elf, J., Li, G.-W. & Xie, X. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194 (2007)
Wang, Y. M., Austin, R. H. & Cox, E. C. Single molecule measurements of repressor protein 1D diffusion on DNA. Phys. Rev. Lett. 97, 048302 (2006)
Studitsky, V. M., Clark, D. J. & Felsenfeld, G. Overcoming a nucleosomal barrier to transcription. Cell 83, 19–27 (1995)
Bonne-Andrea, C., Wong, M. & Alberts, B. In vitro replication through nucleosomes without histone displacement. Nature 343, 719–726 (1990)
Eggleston, A. K., O’Neill, T. E., Bradbury, E. M. & Kowalczykowski, S. C. Unwinding of nucleosomal DNA by a DNA helicase. J. Biol. Chem. 270, 2024–2031 (1995)
Mollazadeh-Beidokhti, L., Deseigne, J., Lacoste, D., Mohammad-Rafiee, F. & Schiessel, H. Stochastic model for nucleosome sliding under an external force. Phys. Rev. E 79, 031922 (2009)
We thank M. Gottesman, R. Gonzalez and members of the Greene laboratory for discussion and assistance throughout this work. We thank P. Modrich for providing an expression construct encoding EcoRIE111Q, R. Landick and K. Adelman for providing RNAP constructs, and J. Gelles for providing plasmids encoding RecBCD. I.J.F. was supported by an NIH Fellowship (F32GM80864). Funding was provided by the National Institutes of Health (GM074739 and GM082848 to E.C.G.). This work was partially supported by the Initiatives in Science and Engineering program through Columbia University, the Nanoscale Science and Engineering Initiative of the National Science Foundation under NSF Award Number CHE-0641523, and by the New York State Office of Science, Technology, and Academic Research. E.C.G is an Early Career Scientist with the Howard Hughes Medical Institute. We apologize to colleagues whose work we were unable to cite owing to length restrictions.
The authors declare no competing financial interests.
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Finkelstein, I., Visnapuu, M. & Greene, E. Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 468, 983–987 (2010) doi:10.1038/nature09561
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