Figure 3.
53BP1’s p53-Regulatory and DSB Repair Activities Are Distinct and Separable
(A) Generation of 53BP1ΔBRCT alleles using CRISPR-Cas9 technology. Top: schematic representation of TP53BP1 gene locus showing the two sgRNA pairs (triangles) used to excise BRCT-encoding exonic sequences. Bottom: immunoblot of lysates prepared from two 53BP1ΔBRCT MCF-7 lines with epitope-specific 53BP1 antibodies showing the expression of mutant 53BP1ΔBRCT protein.
(B) N3 resistance assay was performed as in Figures 1A and 1B. Mean ± SD (n = 2, plated in triplicate).
(C) Subnuclear 53BP1 localization was analyzed by indirect immunofluorescence in indicated cell lines following mock or irradiation (5 Gy, 4 hr) treatments.
(D) The survival of MCF-7 cell lines of indicated genotype following control or X-ray irradiation treatments was assessed by colony survival assay. Mean ± SD (n = 3, plated in triplicate).
(E) The survival of 53BP1Δ cells stably complemented with indicated 53BP1 transgenes following control or X-ray irradiation treatments was assessed as in (D). Mean ± SD (n = 3, plated in triplicate). See also Figure S4.