Supplementary Figure 5: Complete reaction lacking PAXX (A-D) or including a PAXX mutant deficient in interactions with Ku (E-G).

(A) Representative time-trace showing rupture events observed when a complete reaction is assembled in the absence of PAXX. (B) Histogram of observed rupture amplitudes with Gaussian fit to the main peak. A total of 165 events is observed. The maximum of the Gaussian is located at 160 ± 2 nm (SEM; σ = 17 nm; n = 77) (red line). (C) Histogram of synaptic event lifetimes (i.e. with an amplitude contained within three standard deviations of the Gaussian peak) is fitted by a single-exponential distribution (red line), giving a mean duration of 9 ± 1.8 s (SEM, n = 77). (D) Force-dependence of synaptic lifetime in these conditions indicates the lifetime is only very weakly force-dependent. Data were collected using a different preparation of components as in (A-C) but are the same within error. Error bars: SEM (n = 62, n = 63 and n = 53 for low, medium and high force, respectively). (E) Representative time-trace showing rupture events observed when a complete reaction is assembled using the V199A/F201A mutant of PAXX instead of wild-type PAXX. (F) Histogram of observed rupture amplitudes with Gaussian fit to the main peak. A total of 77 events is observed. The maximum of the Gaussian is located at 166 ± 2 nm (SEM; σ = 9 nm; n = 39) (red line). (G) Histogram of synaptic event lifetimes (i.e. with an amplitude contained within three standard deviations of Gaussian peak) is fitted by a single-exponential distribution (red line), giving a mean duration of 5.4 ± 1.3 s (SEM, n = 39). Experiments were carried out on DNA ends lacking phosphate groups.