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. 2012 Oct 10;287(48):40618–40628. doi: 10.1074/jbc.M112.410951

FIGURE 6.

FIGURE 6.

53BP1 inhibits HR similarly to RNF168. A, RNF168 is required for 53BP1 IRIF. Cells were treated with siRNAs and 10 Gy of IR (Cs137) as in Fig. 4A and allowed to recover 4 h prior to fixation and immunostaining with 53BP1 antibodies. Shown are 53BP1 immunostaining signals from representative nuclei from each siRNA treatment. B, 53BP1 inhibits HDR and SSA, and to a lesser extent Alt-EJ. The reporters shown in Fig. 1A were integrated separately into 53BP1−/− MEFs. These cell lines were transfected with an expression vector for I-SceI, along with an expression vector for 53BP1 or a control EV. Shown are the frequencies of GFP+ cells relative to parallel 53BP1−/− samples, normalized to transfection efficiency via a parallel transfection with a GFP expression vector. *, p < 0.0001 (n ≥ 6). Also shown are immunoblot signals for 53BP1 and GAPDH for WT MEFs, 53BP1−/− MEFs, and 53BP1−/− MEFs following transfection with the 53BP1 expression vector. C, expression of a fragment of 53BP1 (dn53BP1) causes a dominant negative disruption of 53BP1 IRIF. Shown is a diagram of 53BP1 and the dn53BP1 fragment that is fused downstream from tandem nuclear localization signals (NLS) and the HA immunotag. U2OS cells were transfected with an expression vector for dn53BP1, cultured for 2 days, treated with 6 Gy of IR (Cs137), and allowed to recover for 4 h prior to fixation and immunostaining. Shown are HA and 53BP1 immunostaining signals for four representative nuclei. The 53BP1 antibody only detects the endogenous protein, because it is directed against the N terminus, which is absent from dn53BP1. D, the peptide dn53BP1 only causes an increase in HDR and SSA in RNF168-proficient cells. U2OS reporter cells were transfected with siCTRL or siRNF168 2 days prior to co-transfection of the respective siRNA and an expression vector for I-SceI, along with an expression vector for dn53BP1 or EV. Shown are the frequencies of GFP+ cells for the HDR and SSA reporter cell lines for each siRNA and expression vector treatment, relative to siCTRL and EV. *, p < 0.0001 (n ≥ 6).