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

Autor: Graves ET; Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France., Duboc C; Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France., Fan J; Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France., Stransky F; Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France., Leroux-Coyau M; Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France., Strick TR; Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
Jazyk: angličtina
Zdroj: Nature structural & molecular biology [Nat Struct Mol Biol] 2015 Jun; Vol. 22 (6), pp. 452-7. Date of Electronic Publication: 2015 May 11.
DOI: 10.1038/nsmb.3019
Abstrakt: 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.
Databáze: MEDLINE
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