Fig. 1. Inactivation of the 53BP1 pathway leads to hyperrecombination for both HR/STGC and BIR/LTGC.

a Schematic drawing of the EGFP-HR/STGC reporter and the repair product after endonuclease I-SceI cleavage (left). The HR frequency was determined in U2OS (EGFP-HR/STGC) cells expressing shRNAs for 53BP1 and RIF1 (middle), or with 53BP1-KO (three KO clones), following infection with lentiviruses encoding I-SceI endonuclease (right). The recombination frequency was determined by FACS analysis, 5 days post-infection. Western blot analysis was conducted to confirm 53BP1 and RIF1 knockdown and 53BP1 KO (Supplementary Fig. 1a). (n = 5 replicates). b Schematic drawing of the EGFP-BIR/LTGC reporter and the repair products using synthesis-dependent strand annealing (SDSA) or end joining (EJ) to complete BIR, termed as BIR-SDSA and BIR-EJ, respectively. The tract length for BIR-SDSA is 3.8 kb, and for BIR-EJ, it ranges from 0.9 kb to 3.8 kb. c U2OS (EGFP-BIR/LTGC) cells, infected with lentiviruses encoding shRNAs for 53BP1 (left) and RIF1 (right) with a vector control, or harboring 53BP1-KO (middle), were assayed for BIR frequency by determining the percentage of EGFP positive cells with FACS analysis 5 days post-infection of I-SceI lentiviruses. The expression of 53BP1 and RIF1 is shown by Western blotting and qPCR, respectively (Supplementary Fig. 1b). (n = 5 replicates). d, e U2OS (EGFP-BIR/LTGC) WT or 53BP1-KO cells stably expressing shRNAs targeting BRCA1, PIF1, POLD3 or RAD51 (d), or RAD52 (e, left), as well as U2OS (EGFP-BIR/LTGC) WT or RAD52-KO cells expressing 53BP1 shRNAs (e, right), were infected with lentiviruses encoding I-SceI, and the percentage of EGFP-positive cells was determined by FACS, 5 days post-infection. The expression of indicated proteins was examined by qPCR (Supplementary Figs. 1c and S14b). (d n = 5 replicates, e n = 4 replicates). Source data are provided as a Source data file.