General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms E. coli Mfd translocase enables transcription-coupled repair (TCR) by displacing RNA polymerase (RNAP) stalled on a DNA lesion and then coordinating assembly of the UvrAB(C) components at the damage site [1, 2, 3, 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 attached [5, 6]. Using a series of single-molecule assays, we find that recruitment of UvrA and UvrAB to Mfd-RNAP arrests the translocating complex and causes its dissolution. Correlative singlemolecule 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 twenty-to two hundred times more strongly to Mfd-RNAP than to DNA damage. These observations provide a quantitative framework for comparison of complementary DNA repair pathways in vivo. 2The conformational changes which take place in Mfd upon docking to, and activation by, stalled RNAP [7, 8, 9] enable it to bind to DNA upstream of RNAP and translocate along DNA against stalled RNAP [10, 11], and to expose a UvrB homology module and recruit UvrA [3]. Remarkably, singlemolecule assays have shown that after displacing stalled RNAP to make the lesion accessible for repair, Mfd continues to translocate slowly and processively with RNAP attached to it [5, 6]. These assays help explain recent results showing that TCR can also accelerate repair of damaged sites downstream of the stall site [12]. Nevertheless, the role of the translocating Mfd-RNAP complex in stimulating repair by UvrAB(C) remains unclear. Here three single-molecule assays based on magnetic trapping [13] are brought to bear on the system.In the tethered-RNAP translocation assay we stall biotinylated RNAP after transcribing twenty bases using only ATP, UTP and GTP on a DNA cassette that lacks C residues. We then tether the RNAP to a streptavidin-coated magnetic bead, and anchor the linear DNA template at one end to a modified glass coverslip. We thus obtain an RNAP stalled ~1 kbp from one end of an ~8 kbp DNA as it transcribes towards the distant glass surface [14] ( Figure 1A). The DNA is extended away from the surface by a vertical force (F = 1 picoNewton, or pN) applied to the bead using a pair of magnets located above the sample, and the bead's position above the surface is detected in real-time using computer-aided videomicroscopy. Addition of 100 nM Mfd and 2 mM ATP causes motion of the bead towards the surface as an Mfd-RNAP complex forms and translocates along the DNA (Fig. 1B) [5, 6]. The complex is Michaelian with respect to ATP, with = 4.7 ± 0.1 bp/s (SEM) and = 16 ± 0.4 µM (SEM) (Extended Data Figure 1). In ~...

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.