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Initiation of transcription-coupled repair characterized at single-molecule resolution

Nature volume 490, pages 431–434 (2012)Cite this article

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Abstract

Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase1,2,3. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

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Figure 1: Experimental approach and real-time dissociation of stalled RD e by Mfd.
Figure 2: Mfd ATP usage and displacement of stalled RNAP.
Figure 3: Kinetic characterization of the action of Mfd on RDe.
Figure 4: Model of RNAP displacement by Mfd during TCR.

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Acknowledgements

We thank K. Neumann for showing us how to perform global fitting with the Igor software package. K.H. was supported by a PhD scholarship from the Frontieres Interdisciplinaires du Vivant Doctoral Program and the Fondation pour la Recherche Médicale. Work in the laboratory of N.J.S. was supported by BBSRC grant BB/I003142/1, and work in the laboratory of S.A.D. was supported by National Institutes of Health grant GM073829. This work was also made possible by a EURYI grant, in addition to CNRS and University of Paris Diderot core funding, to T.R.S.

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Author notes

  1. Kévin Howan, Lars F. Westblade & Sylvain Zorman

    Present address: Present addresses: Statistical Physics Laboratory, Ecole Normale Superieure, 24 rue Lhomond 75005 Paris (K.H.); Department of Pathology and Laboratory Medicine, Division of Infectious Disease Diagnostics, North Shore LIJ Health System Laboratories, 10 Nevada Drive, Lake Success, New York 11718, USA (L.F.W.); Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520, USA (S.Z.).,

Authors and Affiliations

  1. Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité F-75205 Paris, France,

    Kévin Howan, Nicolas Joly, Wilfried Grange, Sylvain Zorman & Terence R. Strick

  2. DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK,

    Abigail J. Smith & Nigel J. Savery

  3. The Rockefeller University, 1230 York Avenue, New York, 10065, New York, USA

    Lars F. Westblade & Seth A. Darst

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Contributions

K.H., N.J.S. and T.R.S. devised and carried out experiments; S.A.D., K.H., N.J., N.J.S., A.J.S., T.R.S. and L.F.W. provided unique reagents and A.J.S. carried out further control experiments; K.H., W.G., N.J.S., T.R.S. and S.Z. analysed data; and S.A.D., N.J.S. and T.R.S. wrote the paper.

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Correspondence to Terence R. Strick.

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This file contains the Supplementary Materials and Methods, Supplementary References, Supplementary Figures 1-12 and the Supplementary Table. (PDF 2633 kb)

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Howan, K., Smith, A., Westblade, L. et al. Initiation of transcription-coupled repair characterized at single-molecule resolution. Nature 490, 431–434 (2012). https://doi.org/10.1038/nature11430

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