Extended Data Fig. 4: RAD52 limits the premature usage of TMEJ in BRCA2 mutants to prevent the formation of chromosomal fusions.
From: POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis
a, 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 = 4 independent experiments). b, γH2AX foci after IR in G1 and G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU− G1 and G2 cells were analysed (n = 2 independent experiments for G1 and n = 3 independent experiments for G2, except siLIG4/siLIG4 + siRAD52/siBRCA2 + siLIG4: n = 2 independent experiments). The G2 data without siLIG4 are also shown in Extended Data Fig. 3e. c, γH2AX foci after IR in G2 fibroblasts lacking XLF. 2BN hTERT (XLF deficient: XLF*) cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU− G2 cells were analysed (n = 3 independent experiments). XLF mutants have much higher levels of unrepaired breaks compared with WT fibroblasts due to their defect in canonical non-homologous end joining, a major pathway for repairing IR-induced DSBs in G2 (compare to Extended Data Fig. 3f). d, Chromatid breaks and fusions after IR and CPT in G2 HeLa cells. Cells were transfected with siRNAs and treated with IR (2 Gy X-rays) or CPT (20 nM, 1 h). Chromosome spreads were obtained by PCC 8-10 h post IR/CPT treatment. Chromatid breaks and fusions were quantified per spread and normalized to 70 chromosomes (n = 4 independent experiments, except siBRCA2 + siPOLθ/siBRCA2 + siRAD52 + siPOLθ: n = 3 independent experiments). Examples of chromosome spreads with breaks (red arrows) and fusions (blue arrows) in irradiated HeLa cells are shown (scale bar, 5 µm). All data show the mean ± s.e.m., except for conditions in (b) with n = 2 independent experiments for which only the mean is shown. Individual experiments, each derived from 30 (G2 spreads) or 40 cells (G1 and G2), are shown as dots. *: P < 0.05; **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.
