In Escherichia coli, the Mfd protein displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Despite extensive in vitro investigation, the molecular mechanistic details of these interactions inside living cells remain unclear. Specifically, the identity of the intermediates orchestrating the handoff of the stall site from the transcription to the repair machineries remains unknown. To investigate the stoichiometry and dynamics of the intermediates formed during this process in real time, we visualized fluorescently-tagged Mfd and UvrA proteins at the single-molecule level in actively growing cells. We determined that Mfd is auto-inhibited in cells, and is recruited to ternary elongation complexes upon transcriptional stress. Further, using mutants of UvrA, we determined the roles of ATP hydrolysis in the formation and disassembly of the handoff complex. We identified that efficient and coordinated disassembly of the handoff complex formed by Mfd-UvrA2-UvrB complexes is contingent upon the ability of UvrB to be loaded at the stall site. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells reveals a ‘facilitated dissociation’ mechanism culminating in the loading of the repair machinery at sites of transcriptional stress.