Fig. 4: Time course analysis of the recruitment of Halo-tagged NHEJ factors to DNA breaks.

A Experimental approach to analyze the chromatin binding of the tagged DNA repair factors over time. B Plot of the static fraction of Halo-Ku70, Halo-DNA-PKcs, and Halo-XRCC4 before and after DNA damage induction (shaded area) with various concentrations of calicheamicin for 3 minutes. Graphs represent the rolling average over three consecutive timepoints (for individual graphs with experimental error see Figure S4A). C Quantification of the timing of the recruitment of Halo-Ku70, Halo-DNA-PKcs, and Halo-XRCC4 after DNA damage induction (N = 3 biological replicates, N = 2 for XRCC4, 120 cells per replicate, Mean ± S.D., one-way ANOVA with Tukey post hoc test, capital letters denote statistically different experimental groups, for Ku70 20 nM vs. 40 nM and 80 nM p = 0.03, 40 nM and 80 nM vs. 120 nM p = 0.01, for DNA-PKcs 20 nM and 40 nM vs. 80 nM p = 0.0001, 80 nM vs. 120 nM p = 0.003, for XRCC4 20 nM vs. 40 nM p = 0.0001, 40 nM vs. 80 nM p = 0.0001). D Quantification of the number of break bound Halo-Ku70 (pooled data for 5 time points after DNA damage induction), Halo-DNA-PKcs (pooled data for 10 time points after DNA damage induction), and Halo-XRCC4 (pooled data for 10 time points after chromatin recruitment) molecules after DNA damage induction with various concentrations of calicheamicin (N = 3 biological replicates, Mean ± S.D., one sided t-test). E Quantification of the DNA repair rate by using static Halo-Ku70 as a marker for the initial number of DNA breaks (D) and the dissociation of XRCC4 (B) to mark the completion of DNA repair by NHEJ (N = 3 biological replicates, Mean ± S.D.). Source data are provided in the Source Data file.