Supplementary Figure 6: Detection of DNA end synapsis by DNA-PKcs and Ku.

The force was switched with 50 s period. (A) Time-trace showing rupture events obtained using 100 pM DNA-PKcs and 10 nM Ku, absent ATP. Red circles and expanded views below highlight transient synaptic events. (B) Amplitude distribution containing 101 events with (red) Gaussian fit to the peak giving the likeliest amplitude 177 ± 8 nm (SD, n = 56). (C) Lifetime distribution of synaptic events (i.e. with an amplitude contained within three standard deviations of the Gaussian peak) follows single-exponential statistics (red line) with mean lifetime 0.1 ± 0.02 s (SEM, n = 56). (D) Time-trace showing rupture events obtained in the presence of 10 nM Ku, absent ATP. Red circles highlight transient, non-specific events. (E) Amplitude distribution containing 133 events with (black) Gaussian fit to the main peak (likeliest amplitude 98 ± 28 nm SD, n = 93) and (red) Gaussian fit to a minor peak (likeliest amplitude 166 ± 6 nm SD, n = 18). Events in the main peak likely correspond to non-specific (tip-scaffold or scaffold-scaffold) interactions; events in the minor peak appear to take place specifically between the DNA ends. Identical scaling to panel B permits direct comparison of results to those obtained when Ku and DNA-PKcs are both present. (F) Single-exponential fit to the lifetime distribution of non-specific events (black line) gives a mean lifetime of 1.2 ± 0.2 s (SEM, n = 93); single-exponential fit to the lifetime distribution of specific events (red line) gives a mean lifetime of 0.7 ± 0.3 s (SEM, n = 18). We conclude that interactions based on Ku alone are distinct from those based on Ku + DNA-PKcs and are for the most part non-specific in nature.