Letter | Published:

Reconstruction of bacterial transcription-coupled repair at single-molecule resolution

Nature volume 536, pages 234237 (11 August 2016) | Download Citation

Abstract

Escherichia coli Mfd translocase enables transcription-coupled repair by displacing RNA polymerase (RNAP) stalled on a DNA lesion and then coordinating assembly of the UvrAB(C) components at the damage site1,2,3,4. Recent studies have shown that after binding to and dislodging stalled RNAP, Mfd remains on the DNA in the form of a stable, slowly translocating complex with evicted RNAP attached5,6. Here we find, using a series of single-molecule assays, that recruitment of UvrA and UvrAB to Mfd–RNAP arrests the translocating complex and causes its dissolution. Correlative single-molecule nanomanipulation and fluorescence measurements show that dissolution of the complex leads to loss of both RNAP and Mfd. Subsequent DNA incision by UvrC is faster than when only UvrAB(C) are available, in part because UvrAB binds 20–200 times more strongly to Mfd–RNAP than to DNA damage. These observations provide a quantitative framework for comparing complementary DNA repair pathways in vivo.

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Acknowledgements

This work was made possible by a China Scholarship Council award to J.F., as well as grants from the French Agence Nationale pour la Recherche (RepOne) and the European Science Foundation (EURYI) to T.R.S., as well as core funding from the CNRS and the University of Paris Diderot. The Strick laboratory is also part of the Programme Equipe Labellisées of the Ligue Contre le Cancer. We thank N. Joly for assistance with protein purification and the Strick laboratory for feedback.

Author information

Affiliations

  1. Institut Jacques Monod, CNRS, UMR7592, University Paris Diderot, Sorbonne Paris Cité F-75205 Paris, France

    • Jun Fan
    • , Mathieu Leroux-Coyau
    •  & Terence R. Strick
  2. DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK

    • Nigel J. Savery
  3. Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS, Inserm, Ecole Normale Supérieure, PSL Research University, F-75005 Paris, France

    • Terence R. Strick
  4. Programme Equipe Labellisées, Ligue Contre le Cancer, 75013 Paris, France

    • Terence R. Strick

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Contributions

J.F., M.L.C., N.J.S. and T.R.S. planned experiments; J.F., M.L.C. and T.R.S. prepared reagents; J.F. performed tethered-RNAP, tethered-DNA and NanoCOSM assays; M.L.C. and T.R.S. performed tethered-RNAP assays. J.F., M.L.C. and T.R.S. conducted data analysis, and N.J.S. and T.R.S. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Terence R. Strick.

Reviewer Information

Nature thanks S. Deindl, J. Elf and J. Roberts for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature19080

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