In the model organism, Escherichia coli, helix distorting lesions are recognized either by the UvrAB damage surveillance complex or by RNA polymerases reading template DNA. Damage recognition in both sub-pathways of nucleotide excision repair is ultimately mediated by the UvrA protein, culminating in the loading of the damage verification enzyme UvrB. We set out to characterize how stalled RNA polymerases are recognized by the transcription repair coupling factor (Mfd), and the stall site is handed over to the nucleotide excision repair machinery (UvrAB) in live cells. We applied single-molecule imaging techniques to interrogate the binding of fluorescently tagged repair factors (Mfd, UvrA and UvrB) in live E coli cells. In combination with a series of structural mutants of Mfd, UvrA and UvrB in repair deficient strains, we have determined the residence time of each of these factors in the various sub-pathways of nucleotide excision repair. These studies have enabled us to identify the composition of the hand-off complex, and the catalytic determinants of successful hand-over of the stall site during the transcription coupled repair reaction. These studies enable us to bridge the gap between existing models built using in vitro experiments and test them in the physiological context of live cells.