Supplementary Figure 4: Effect of downstream components alone.

(A) XRCC4, XLF and ligase IV on their own do not affect the DNA scaffold in standard experimental conditions. (B, C) Low and high concentrations of XRCC4 and XLF do not affect the DNA scaffold in standard experimental conditions. (D, E) Low and high concentrations of XRCC4 and XLF do not affect the DNA scaffold in “high salt” buffer composed of 20 mM Tris-HCl pH 7.6, 150 mM KCl, 1 mM DTT, 0.05% Tween-20 and 0.5 mg/ml BSA. (F, G) Low and high concentrations of XRCC4 and XLF provoke nonspecific compaction of the DNA scaffold in “low salt” buffer composed of 20 mM Tris-HCl pH 7.6, 25 mM KCl, 1 mM DTT, 0.05% Tween-20 and 0.5 mg/ml BSA. (H, I) Low and high concentrations of XRCC4 and XLF provoke nonspecific compaction of regular DNA molecules in buffer used for (F,G). (J,K) The quaternary combination of PAXX, XRCC4, XLF and ligase IV results in non-specific compaction of the DNA scaffold in standard experimental conditions. The absence of such non-specific compaction when Ku is additionally present (i.e. when all components are present but for DNA-PKcs, see Supp. Figure 3I) underscores the role of Ku in regulating non-specific interactions between downstream components and DNA.