Fig. S6.

Kinetics of Rep mutants on target DNA measured using a single-molecule PIFE assay. (A) Structures of Cy3 dye undergoing cis–trans isomerization. Changes in the external environmental, such as the approach of proteins or an increase in solvent viscosity, can hinder the isomer conversion from the trans (fluorescent) state (Upper) to the cis (nonfluorescent) state (Lower), resulting in an increase in fluorescence intensity. (B) Fluorescence spectra of Cy3 on 50 nM target DNA (Table S3) in the absence (red) and presence (blue) of the Rep V69R mutant (500 nM). (C) Relative enhancement in the fluorescence intensity with various Rep mutants. The enhanced intensity was calculated as the ratio of the total integrated intensity of each spectrum. (D) Representative single-molecule time trajectory of Cy3 upon the injection of the V69R mutant (20 nM). The Rep-bound state of the target DNA (on-state) shows a higher level of fluorescence emission from Cy3, whereas the lower level of emission represents free DNA without Rep binding (off-state). The black arrow indicates the time when Rep mutants were injected into a flow chamber. (E) Histograms for the dwell time of the Rep on-state (Left) and Rep off-state (Right), with single exponential fits (red curves).