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.

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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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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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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.

Correspondence to Terence R. Strick.

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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).

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