U2OS-based lines were maintained under standard conditions. cDNA cloning was by standard procedures. siRNA transfections were with Lipofectamine RNAiMAX (Invitrogen). IR was administered with a Faxitron X-ray machine (Faxitron X-ray Corporation). ATM inhibition was by KU-55933 (KuDOS Pharmaceuticals). Laser micro-irradiation was with a FluoView 1000 confocal microscope (Olympus) with 37°C heating stage (Ibidi) and 405 nm diode (6 mW). FRAP was performed when laser-track accumulation of GFP-tagged protein reached maximal steady-state level. For immunofluorescence, cells were pre-extracted or not, fixed with 2% paraformaldehyde, permeabilized and stained. For whole cell extracts, cells were lysed on plates with 2% SDS, 50 mM Tris-HCl pH 7.5, 20 mM N-ethylmaleimide (Sigma-Aldrich) and protease inhibitor cocktail (Roche). To immunoprecipitate 53BP1, BRCA1 and sumoylated proteins, different lysis and binding buffers were used (Supplementary Information). HR and NHEJ assays were as previously described17 (link),28 (link). For IR survival, cells were transfected with siRNA and exposed to IR. After 10-14 days, colonies were stained with 0.5% crystal violet/20% ethanol, counted and normalized to plating efficiencies. For Florescence-Activated Cell Sorting (FACS) of propidium iodide-stained cells, data were analyzed by FlowJo software. All error-bars represent STDEV. Detailed descriptions of methods are provided in Supplementary Information.