Supplementary Figure 3: DNA end synapsis in NHEJ reactions lacking one or two components or employing a mutant component.

The stated component is withheld from a “complete” reaction. Panels display (left) a time-trace, (middle) an amplitude distribution with Gaussian fit to the peak (red), and (right) a lifetime distribution for synaptic events with exponential fit (red). The number of events in the lifetime fits is equal to the number of events in the Gaussian fit and is a subset of the total number of events observed. DNA ends are phosphorylated (P + ) or unphosphorylated (P-). Errors are SEM. For (C, F) the coiled-coil domain of XRCC4 is present. (a) Reaction lacking ligase IV (P-). 46 events were observed, with likeliest amplitude 161 ± 4 nm and mean lifetime 4.4 ± 1.6 s (n = 25). (b) Reaction lacking XLF (P-). 179 events were observed, with likeliest amplitude 160 ± 2 nm and mean lifetime 3.2 ± 0.6 s (n = 74). (c) Reaction lacking XRCC4 NTD/CTD (P-). 137 events were observed, with likeliest amplitude 165 ± 2 nm and mean lifetime 2.1 ± 0.4 s (n = 80). (d) Reaction lacking ligase IV (P + ). 79 events were observed, with likeliest amplitude 157 ± 2 nm and mean lifetime 3.3 ± 0.7 s (n = 47). (e) Reaction lacking XLF (P + ). 135 events were observed, with likeliest amplitude 159 ± 2 nm and mean lifetime 3 ± 0.5 s (n = 78). (f) Reaction lacking XRCC4 NTD/CTD (P + ). 94 events were observed, with likeliest amplitude 158 ± 2 nm and mean lifetime 2.7 ± 0.7 s (n = 49). (g) Reaction with catalytically dead ligase IV (P + ). 181 events were observed, with likeliest amplitude 163 ± 2 nm and mean lifetime 34.5 ± 6.9 s (n = 82). (h) Reaction with mutant XLF (P-). 132 events were observed, with likeliest amplitude 164 ± 1 nm and mean lifetime 44 ± 12.6 s (n = 84). (i) Reaction lacking DNA-PKcs (P + ). Time-trace shows no synapsis in these conditions. (j) Reaction lacking XLF and PAXX (P-). Time-trace shows no synapsis in these conditions.