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A dynamic DNA-repair complex observed by correlative single-molecule nanomanipulation and fluorescence

Nature Structural & Molecular Biology volume 22, pages 452457 (2015) | Download Citation

Abstract

We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at 4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.

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Acknowledgements

We thank N. Savery, S. Darst, R. Landick and J. Gelles for stimulating conversations, members of the Strick laboratory for critical discussions, N. Joly (Institut Jacques Monod) for a generous gift of Mfd and W. Grange (University of Paris Diderot) for cloning of the AviTag into the β′ subunit of RNAP. This work was made possible by a European Science Foundation (EURYI) grant, European Union Seventh Framework Program grant (HEALTH-F4-2008-223545) and a French Agence National de la Recherche grant (RepOne) to T.R.S., in addition to support from the Fondation Fourmentin-Guilbert, and CNRS and University of Paris Diderot core funding.

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Author notes

    • Evan T Graves

    Present Address: Ventana Medical Systems, Oro Valley, Arizona, USA.

Affiliations

  1. Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.

    • Evan T Graves
    • , Camille Duboc
    • , Jun Fan
    • , François Stransky
    • , Mathieu Leroux-Coyau
    •  & Terence R Strick

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Contributions

T.R.S. and E.T.G. devised and built the experimental system; E.T.G., C.D., J.F., F.S., M.L.-C. and T.R.S. purified protein and nucleic acid reagents and carried out experiments and analysis; T.R.S. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Terence R Strick.

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https://doi.org/10.1038/nsmb.3019

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