Figure 4.
A single-molecule dye-quenching assay for XPD translocation along replication protein A (RPA)-coated DNA. (a) A DNA oligonucleotide is immobilized on the surface of a passivated flowcell and decorated with Cy5-labeled RPA. The FeS cluster of XPD helicase quenches Cy5 fluorescence in a distance-dependent manner. (b) When ATP is added, XPD translocation toward RPA initially quenches the Cy5 fluorescence (arrow 1), and eventually XPD bypasses the RPA (arrow 2), which is revealed as an eventual recovery of the Cy5 intensity. (c) Examples of intensity trajectories showing XPD approaching (arrow 1) and moving past (arrow 2) RPA.