Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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


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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others


  1. Selby, C. P. & Sancar, A. Molecular mechanism of transcription-repair coupling. Science 260, 53–58 (1993)

    Article  CAS  ADS  Google Scholar 

  2. Witkin, E. M. Time, temperature, and protein synthesis: a study of ultraviolet-induced mutation in bacteria. Cold Spring Harb. Symp. Quant. Biol. 21, 123–140 (1956)

    Article  CAS  Google Scholar 

  3. Mellon, I. & Hanawalt, P. C. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature 342, 95–98 (1989)

    Article  CAS  ADS  Google Scholar 

  4. Hanawalt, P. C. & Spivak, G. Transcription-coupled DNA repair: two decades of progress and surprises. Nature Rev. Mol. Cell Biol. 9, 958–970 (2008)

    Article  CAS  Google Scholar 

  5. Selby, C. P. & Sancar, A. Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor. Proc. Natl Acad. Sci. USA 88, 8232–8236 (1991)

    Article  CAS  ADS  Google Scholar 

  6. Selby, C. P. & Sancar, A. Transcription-repair coupling and mutation frequency decline. J. Bacteriol. 175, 7509–7514 (1993)

    Article  CAS  Google Scholar 

  7. Park, J. S., Marr, M. T. & Roberts, J. W. E. coli transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation. Cell 109, 757–767 (2002)

    Article  CAS  Google Scholar 

  8. Smith, A. J., Szczelkun, M. D. & Savery, N. J. Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase. Nucleic Acids Res. 35, 1802–1811 (2007)

    Article  CAS  Google Scholar 

  9. Smith, A. J. & Savery, N. J. Effects of the bacterial transcription-repair coupling factor during transcription of DNA containing non-bulky lesions. DNA Repair (Amst.) 7, 1670–1679 (2008)

    Article  CAS  Google Scholar 

  10. Chambers, A. L., Smith, A. J. & Savery, N. J. A DNA translocation motif in the bacterial transcription–repair coupling factor, Mfd. Nucleic Acids Res. 31, 6409–6418 (2003)

    Article  CAS  Google Scholar 

  11. Deaconescu, A. M. et al. Structural basis for bacterial transcription-coupled DNA repair. Cell 124, 507–520 (2006)

    Article  CAS  Google Scholar 

  12. Deaconescu, A. M. & Darst, S. A. Crystallization and preliminary structure determination of Escherichia coli Mfd, the transcription-repair coupling factor. Acta Crystallogr. F 61, 1062–1064 (2005)

    Article  CAS  Google Scholar 

  13. Westblade, L. F. et al. Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction. Nucleic Acids Res. 38, 8357–8369 (2010)

    Article  CAS  Google Scholar 

  14. Deaconescu, A. M., Savery, N. J. & Darst, S. A. The bacterial transcription repair coupling factor. Curr. Opin. Struct. Biol. 17, 96–102 (2007)

    Article  CAS  Google Scholar 

  15. Park, J. S. & Roberts, J. W. Role of DNA bubble rewinding in enzymatic transcription termination. Proc. Natl Acad. Sci. USA 103, 4870–4875 (2006)

    Article  CAS  ADS  Google Scholar 

  16. Savery, N. J. The molecular mechanism of transcription-coupled DNA repair. Trends Microbiol. 15, 326–333 (2007)

    Article  CAS  Google Scholar 

  17. Revyakin, A., Liu, C. Y., Ebright, R. H. & Strick, T. R. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139–1143 (2006)

    Article  CAS  ADS  Google Scholar 

  18. Revyakin, A., Ebright, R. H. & Strick, T. R. Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation. Proc. Natl Acad. Sci. USA 101, 4776–4780 (2004)

    Article  CAS  ADS  Google Scholar 

  19. Revyakin, A. et al. Single-molecule DNA nanomanipulation: detection of promoter-unwinding events by RNA polymerase. Methods Enzymol. 370, 577–598 (2003)

    Article  CAS  Google Scholar 

  20. Smith, A. J. & Savery, N. J. RNA polymerase mutants defective in the initiation of transcription-coupled DNA repair. Nucleic Acids Res. 33, 755–764 (2005)

    Article  CAS  Google Scholar 

  21. Kou, S. C. et al. Single-molecule Michaelis–Menten equations. J. Phys. Chem. B 109, 19068–19081 (2005)

    Article  CAS  Google Scholar 

  22. Brueckner, F., Hennecke, U., Carell, T. & Cramer, P. CPD damage recognition by transcribing RNA polymerase II. Science 315, 859–862 (2007)

    Article  CAS  ADS  Google Scholar 

  23. Komissarova, N. et al. Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell 10, 1151–1162 (2002)

    Article  CAS  Google Scholar 

  24. Srivastava, D. B. & Darst, S. A. Derepression of bacterial transcription-repair coupling factor is associated with a profound conformational change. J. Mol. Biol. 406, 275–284 (2011)

    Article  CAS  Google Scholar 

  25. Landick, R. The regulatory roles and mechanism of transcriptional pausing. Biochem. Soc. Trans. 34, 1062–1066 (2006)

    Article  CAS  Google Scholar 

  26. Manelyte, L. et al. Regulation and rate enhancement during transcription-coupled DNA repair. Mol. Cell 40, 714–724 (2010)

    Article  CAS  Google Scholar 

  27. Selby, C. P. & Sancar, A. Structure and function of transcription-repair coupling factor. I. Structural domains and binding properties. J. Biol. Chem. 270, 4882–4889 (1995)

    Article  CAS  Google Scholar 

  28. Deaconescu, A. M., Sevostyanova, A., Artsimovitch, I. & Grigorieff, N. Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface. Proc. Natl Acad. Sci. USA 109, 3353–3358 (2012)

    Article  CAS  ADS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Terence R. Strick.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing