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Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase

Abstract

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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Figure 1: RecBCD removes RNAP from DNA.
Figure 2: Disruption of EcoRI E111Q and lac repressor by RecBCD.
Figure 3: Nucleosomes can be pushed along DNA.
Figure 4: Protein displacement mechanisms.

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Acknowledgements

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.

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Authors

Contributions

I.J.F. did all cloning and ensemble-level biochemical characterization, and conducted and analysed RecBCD collision experiments with RNAP, EcoRIE111Q and LacI. M.-L.V. conducted and analysed RecBCD collision experiments with nucleosomes. I.J.F., M.-L.V. and E.C.G. discussed the data and co-wrote the paper.

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Correspondence to Eric C. Greene.

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The authors declare no competing financial interests.

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The file contains Supplementary Materials and Methods, Supplementary Figures 1-9 with legends, Supplementary Tables 1-2 and additional references. (PDF 2320 kb)

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Finkelstein, I., Visnapuu, ML. & Greene, E. Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 468, 983–987 (2010). https://doi.org/10.1038/nature09561

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