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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 site1-4. 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., The conformational changes that take place in Mfd upon docking to, and activation by, stalled RNAP (7-9) enable it to bind to DNA upstream of RNAP and translocate along DNA [...]

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 [...]

Author(s): Terence R. Strick (corresponding author); Vincent Croquette; David Bensimon Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a [...]

Focuses on a single-molecule analysis of DNA uncoiling induced by a type II topoisomerase. Use of a single Drosophila melanogaster topoisomerase II; Monitoring of DNA extension in the presence of adenosine triphosphate; Relaxation of two supercoils during a single catalytic turnover.