Figure 1. Characterization of a separation of function mutant 53BP1.

(A) Top: Representative flow cytometry plots measuring CSR after stimulation of WT and BRCA1^−/−53BP1^−/− B cells infected with retroviruses expressing 53BP1^DB (amino acids 1-1710), the N-terminal mutant 53BP1^8A or empty vector (EV). Numbers represent the percentages of IgG1 switched cells. B220 is a B cell marker. Bottom: Dot plot indicating IgG1 CSR as a percentage of WT value in the same experiment. Three independent experiments are shown. **: p<0.001 (two-tailed unpaired t-test); BRCA1/53BP1+DB vs. BRCA1/53BP1+8A, p>0.1 which is not significant (ns). (B) BRCA1^−/−53BP1^−/− B cells were reconstituted with empty vector, 53BP1^DB and 53BP1^8A retroviruses and treated with PARPi. The arrows indicate representative images of aberrant chromosomes. Dot plot indicates the total aberrations per cell in three independent experiments. At least 50 metaphases were analyzed for each genotype in each experiment. **: p<0.01 (two-tailed unpaired t-test);ns: not significant. (C) Western blot analysis of 53BP1 expression in WT B cells and BRCA1^−/−53BP1^−/− B cells stimulated and infected with empty vector, 53BP1^DB or 53BP1^8A. (D) BRCA1^−/−53BP1^−/− B cells infected with EV, 53BP1^DB or 53BP1^8A retroviruses were assayed for IRIF (10 Gy, 2 hour recovery) for RIF1 (red, top panel) and 53BP1 (red, lower panel). Cells were counterstained with DAPI (blue). Scale bar represents 10 μm. See also Figure S1.