Figure 4. SSB binding modes and diffusion mechanism.

(A) Schematic of fluorescently labeled SSB, SSB_f, ssDNA wrapping experiment. A Cy5-labeled DNA construct is tethered between two optical traps under a constant tension of 5 pN. Upon binding of an AlexaFluor555-labeled SSB, both DNA extension change, Δx, and single-molecule FRET are measured simultaneously. (B) Scatter plot of FRET efficiency and Δx. Data (circles) are assigned to 4 states (red (i), blue (ii), black (iii), and green (iv)) based on the value of FRET and Δx. A density map of the combined FRET-extension states overlaid with the scatter plot confirms that the data can be separated into 4 states. Cartoon illustrations of nucleoprotein complexes demonstrate possible SSB wrapping configurations corresponding to the 4 assigned states. (C) Representative traces showing combined fluorescence and DNA extension measurements. Change in extension (top; boxcar averaged to 50 Hz) and fluorescence (middle; boxcar averaged to 0.5 Hz) of donor (SSB_f, green) and acceptor (Cy5, red) are measured simultaneously. Together, FRET efficiency (bottom; blue) and extension change (top; black) reveal the SSB wrapping states (i and ii, iii and iv) and their dynamics (ssDNA wrapping/releasing and sliding).

DOI: http://dx.doi.org/10.7554/eLife.08193.016

Figure 4—figure supplement 1. Mechanism of SSB diffusion.

Cartoon illustrations of nucleoprotein complexes diffusing along ssDNA with different proposed mechanisms. Schematic FRET efficiency and Δx displaying multiple transitions between states (i, ii, iii, iv). In a sliding or reptation mechanism, FRET transitions occur independently of changes in wrapping state (top panel). A rolling mechanism involves SSB displacement by wrapping one end of DNA followed by releasing the other (bottom panel; i → iii → ii or ii → iii → i). No examples (0 of N = 82) of rolling are observed in our experiment.

DOI: http://dx.doi.org/10.7554/eLife.08193.017