Initiation of transcription-coupled repair characterized at single-molecule resolution

Transcription-coupled repair employs 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 [1–3]. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of E. coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalyzing two irreversible, ATP-dependent steps with different structural, kinetic, and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could serve 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.