Figure 3.

The C-terminal arms of Ku are required for recruitment of LigD and full stabilization of the pre-ligation complex. EMSA gel of a 50 bp blunt-ended DNA substrate incubated with 0.2 μM (A) Ku_wt and (B) Ku_core and an increasing concentration of LigD (0–0.8 μM, 0.1 μM increments). The arrows indicate the number of Ku homodimers loaded on the DNA substrate_. (C) Time-trace of molecular forceps experiment (blue), where a blunt-ended DNA substrate is used together with Ku_wt (5 nM) and LigD (5 nM). End-to-end synapses were detected upon repeated force cycling (red). (D) Lifetime distribution of the specific end-binding events is fit to a single-exponential, giving a mean lifetime of 18.5 ± 1.2 s (SEM, n = 459). End-specific events are identified (inset) as having a Δl value within three standard deviations of the mean expected amplitude change given bridge mechanics (Gaussian fit in red, <Δl> = 138 ± 15 nm, SD, n = 486). (E, F) as C and D but for Ku_core, giving a mean synapsis lifetime of 5.1 ± 0.4s (SEM, n = 270). End-specific events are identified (inset) as having a Δl value within 3 standard deviations of the mean expected amplitude change given bridge mechanics (Gaussian fit in red, <Δl> = 126 ± 16 nm, SD, n = 305). (G) Ligation of a 1000 bp blunt-ended DNA substrate with a constant amount of LigD (0.1 μM) and increasing concentration of Ku_wt (0–1 μM, 0.2 μM increments). The grey area in the LigD bar indicates the absence of LigD in the corresponding sample, where only Ku is added at a concentration of 1 μM.