Figure 3. Three color FRET observations support RecA filament sliding.
(A) A schematic of the single molecule three color FRET assay to measure RecA filament sliding. ssDNA (Lfilament = 99 nt) labeled with two acceptor fluorophores (Cy5-red and Cy7-black) with a separation of 33 nt between the fluorophores, was immobilized on the surface. Upon docking of non-homologous donor (Cy3) labeled dsDNA to the pre-formed RecA filament formation, sliding predicts anticorrelated emissions between the two acceptors. (B) Single molecule time traces of Cy3 (green), Cy5 (red) and Cy7 (black) intensities (top panel). Corresponding FRET time traces of FRET between Cy3 and Cy5 (ECy3-Cy5-blue) and FRET between Cy3 and Cy7 (ECy3-Cy7-grey). (C) Normalized cross correlation plot of ECy3-Cy5 and ECy3-Cy7 averaged over 30 molecules and a single exponential fit of the data is overlaid (black). (D). Scatter plot of ECy3-Cy5 and ECy3-Cy7 for 30 molecules showing unique high FRET regions along both axes.