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 
transcription-coupled repair is initiated when RNA polymerase stalled at a DNA 
lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use 
single-molecule DNA nanomanipulation to observe the dynamic interactions of 
Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a 
cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by 
catalysing two irreversible, ATP-dependent transitions with different 
structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a 
long-lived complex that could act as a marker for sites of DNA damage, directing 
assembly of subsequent DNA repair factors. These results provide a framework for 
considering the kinetics of transcription-coupled repair in vivo, and open the 
way to reconstruction of complete DNA repair pathways at single-molecule 
resolution.