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. 2017 Feb 17;292(16):6461–6467. doi: 10.1074/jbc.M117.777474

Figure 4.

Figure 4.

TIRR participates in 53BP1-dependent DNA damage response. Loss of TIRR renders BRCA1-deficient cells resistant to PARP inhibition (A). HeLa cells were transfected with indicated gRNAs and treated with 1 μm PARPi (Olaparib). Colony formation was quantified relative to colonies formed in untreated cells from the same setting. Data are represented as the mean ± S.E. (n = 3). *, p < 0.05. TIRR prevents end resection and RAD51-dependent HR repair in the absence of BRCA1 (B and C). HeLa cells were transfected with indicated gRNAs. At 3 h post-irradiation (10 Gy), cells were processed for RAD51 (B) or RPA (C) immunofluorescence staining. RAD51 (B) or RPA (C) foci were quantified (at least 400 cells were counted for each experiment). Data are represented as the mean ± S.E. (n = 3). *, p < 0.05. 53BP1-TIRR interaction is important for cellular response to PARPi (D). TIRR knock-out MCF10A cells were reconstituted with gRNA-resistant wild-type or Nudix motif deletion mutant of SFB-tagged TIRR. These cells were then infected with indicated control or BRCA1 gRNAs and treated with 1 μm PARPi. Colony formation was quantified relative to colonies formed in untreated cells from the same setting. Data are represented as the mean ± S.E. (n = 3). *, p < 0.05. TIRR functions in DNA repair via its ability to regulate 53BP1 expression (E). Control cells or TIRR knock-out cells with or without 53BP1 overexpression were treated with control or BRCA1 siRNA. Cells were treated with olaparib at various concentrations for 6 days, and viable cell count was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. *, p < 0.05. TIRR is required for cell survival following ionizing radiation (F). HeLa cells transfected with control siRNA (siCTR) or TIRR specific siRNAs (siTIRR#1 and siTIRR#2) were mock-treated or irradiated. Cell survival following irradiation was measured by clonogenic assay according to the “Experimental procedures.” Western blotting analysis was performed to verify the depletion of TIRR following siRNA transfection (lower panel). *, p < 0.05. G, revised model of TIRR-53BP1 interaction and its dissociation in regulating DNA repair and cellular response to PARPi following DNA damage.