Extended Data Fig. 3: RAD52 locates to resected DSBs to prevent their repair in G2-phase BRCA2 mutants.
From: POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis
a, Diagram of the GFP–RAD52 protein: GFP tag (green), RAD52 self-binding domain (orange), RPA-binding domain (purple), RAD51-binding domain (yellow) and nuclear localization signal (NLS, red). Expression levels of endogenous vs. tagged RAD52 were assessed by immunoblotting. GAPDH was used as loading control. The example immunofluorescence (IF) image shows the variability in GFP–RAD52 expression between different cells (scale bar, 10 µm). For foci analysis, cells with very high or very low GFP–RAD52 expression were excluded. b, Kinetics of γH2AX, RAD51 and GFP–RAD52 foci in G2 HeLa cells stably expressing GFP–RAD52. Cells were labelled with EdU, irradiated with 2 Gy X-rays and EdU− G2 cells were analysed (n = 5 independent experiments, except 0.25 h and 5 h: n = 3 or n = 2 independent experiments, respectively). c, Co-localization studies of RAD52, RAD51, pRPA and γH2AX in G2 HeLa GFP–RAD52 cells. Cells were labelled with EdU, irradiated with 2 Gy X-rays and EdU− G2 cells were analysed (n = 2 independent experiments, except co-localization of RAD52/RAD51: n = 3 independent experiments). Example IF images are shown in the right panel (scale bar, 5 µm). d, Spontaneous γH2AX foci in S/G2 HeLa cells. Cells were transfected with siRNAs, labelled as in Fig. 1a and EdU−BrdU+ cells were analysed (n = 4 independent experiments). e, γH2AX foci after IR in G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU− G2 cells were analysed (n = 3 independent experiments). f, γH2AX foci after IR in G2 fibroblast cells. Experiment was performed as in (e), but using 82-6 hTERT (WT) cells (n = 3 independent experiments). Data without siBRCA2 are the same as in Fig. 3e. All data show the mean ± s.e.m, except for (b) and the conditions in (c) with n = 2 independent experiments for which only the mean is shown. Individual experiments, each derived from 40 cells (except (c): 20 cells), are shown as dots. *: P < 0.05; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.
