Fig 2: Effects of resection factors on foci pairing.(a) Frequency of paired 53BP1 foci in MRE11-depleted cells (G1 phase). BJ-hTERT cells were transfected with the indicated siRNA, and after 3 days the cells were irradiated with 2 Gy ionizing radiation and then fixed at the indicated time points. Plotted are the percentages of paired foci among the total foci in individual cells. Statistical analyses were performed using the two-tailed Mann-Whitney test (alpha = 0.05). Red bars represent means. (b) Confirmation of MRE11 depletion by western blotting (cropped blot). (c) Frequency of paired 53BP1 foci in CtIP-depleted cells (G1 phase). Transfection of siRNA, sample preparation, examination of foci pairing, and statistical analyses were performed as described for the MRE11-depleted cells in Fig. 4a. (d) Confirmation of CtIP depletion by western blotting (cropped blot). Full-length blots for Fig. 4b and d are shown in Supplementary Figure S7a,b and S7c,d, respectively.

Fig 3: A proposed model for the role of Abraxas/BRCA1-A complex.Abraxas/BRCA1-A complex inhibits DNA end resection and limits BIR by restricting SLX4/MUS81 chromatin loading through counteracting K63-linked ubiquitin modification catalyzed by RNF168/Ubc13 in response to CPT. In the absence of Abraxas, increased K63-linked ubiquitin conjugation leads to SLX4/MUS81 overloading on chromatin, generating increased seDSBs, which are further processed by MRE11 endonucleases, CTIP and DNA2/BLM, forming an extensive length of ssDNA. Uncontrolled MUS81 loading and excessive end resection also involve RAD52- and POLD3-dependent BIR that results in increased mitotic DNA synthesis, extensive chromosome aberrations, and cell lethality in Abraxas-deficient cell.

Fig 4: CtIP protein level is reduced in Aquarius-depleted cells. (a) Downregulation of CtIP protein level in AQR-knockdown cells. siRNAs against AQR were transfected into five different cell lines, and the cells were then lysed and CtIP and Aquarius were analysed by western blotting 48 h after siRNA transfection. (b) Partial rescue of CtIP by AQR add-back. Aquarius-depleted HCT116 cells transiently expressed GFP (EV) or GFP-tagged Aquarius (WT or HD), and CtIP protein was analysed by western blotting. (c) Analysis of CtIP mRNA by RT-PCR. Total RNA was isolated from HCT116 cells transfected with control siRNA or AQR siRNA, and PCR was performed with appropriate sets of primers after reverse transcription. RT-PCR against β-actin (ACTB) was performed as a loading control. (d) Protein stability of CtIP in AQR-knockdown cells. U2OS and HCT116 cells were treated with CHX (20 μg/ml) and MG-132 (MG, 10 μM) for the indicated periods 48 h after control siRNA or AQR siRNA transfection. Aquarius and CtIP protein levels were analysed by western blotting. Protein levels of CtIP were normalized by β-actin and the relative protein levels of CtIP are shown as a line graph. Data represent mean ± SD from at least three independent experiments. (e and f) Percentage of 53BP1 foci (>5)- and Rad51 foci (>10)-positive cells in AQR-knockdown HeLa (e) and U2OS (f) cells in response to MMC (200 ng/ml, 4 h) or NCS (20 ng/ml, 4 h). (g) Percentage of Rad51 foci (>10)-positive cells in CtIP overproduced AQR-knockdown cells. HCT116 cells, depleted for Aquarius, transiently expressed GFP (EV) or GFP-tagged CtIP (GFP-CtIP), and were immunostained with anti-Rad51 antibody after NCS (20 ng/ml, 4 h) treatment. Data represent mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005.

Fig 5: MRE11 and TDP2 repair TOP3Accs in parallel pathways.a Epistatic relationship between SPRTN and TDP2. Left panel: representative slot-blot. WT and TDP2-/- HCT116 cells were transfected with the indicated TOP3A plasmid constructs for 48 h and co-transfected with either siControl or siSPRTN for 72 h. TOP3Accs were detected by RADAR assay with anti-TOP3A antibody. Right panel: Quantitation of TOP3Accs from three independent RADAR assays as shown in left panel. NT; mock transfected. Error bar indicates mean ± SD. Ordinary one-way ANOVA with Dunnett multiple comparison test. **Adjusted p-value = 0.0083 (Wild-type vs TDP2-/-), **Adjusted p-value = 0.0013 (Wild-type vs siSPRTN), ***Adjusted p-value = 0.0009 (Wild-type vs siSPRTN/TDP2-/-). b Representative images of colony formation assays in indicated genotypes of HCT116 cells after transfection with TOP3A-R364W. Lower panel: quantification of the data from experiments as shown in upper panel. Clonogenic survival histogram data of each cell types were presented after normalized with their respective colony formation efficiency in TOP3A-WT transfection condition. Error bar represents data mean ± SD (n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett multiple comparison test. ****Adjusted p-value = <0.0001 (Wild-type vs TDP2-/-, Wild-type vs siSPRTN, Wild-type vs siSPRTN/TDP2-/-). c Model for the coordinated processing of TOP3Accs by SPRTN and TDP2. d Additive effects of MRE11 and TDP2 on TOP3Accs. WT and TDP2-/- HCT116 cells were transfected with the indicated TOP3A plasmid constructs for 48 h and co-transfected with either siControl or siMRE11 for 72 h. Representative slot blot of RADAR assay samples probed with anti-TOP3A antibody. Quantitation is shown to the right of slot-blot images. Error bar indicates the mean value ± SD (n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett multiple comparison test. *Adjusted p-value = 0.0380 (Wild-type vs siMRE11), *Adjusted p-value = 0.0324 (Wild-type vs TDP2-/-), ****Adjusted p-value = <0.0001 (Wild-type vs siSPRTN/TDP2-/-). e Representative images of colony formation assays in indicated genotypes of HCT116 cells after transfection with TOP3A-R364W. Lower panel: quantification of the data from experiments as shown in upper panel. Clonogenic survival histogram data of each cell types were presented after normalized with their respective colony formation efficiency in TOP3A-WT transfection condition. Error bar represents data mean ± SD (n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett multiple comparisons test. ****Adjusted p-value = <0.0001 (Wild-type vs siMRE11, Wild-type vs TDP2-/-, Wild-type vs siMRE11/TDP2-/-). f, g Effects of ATM, CtIP and TDP2 on TOP3Accs. WT and TDP2-/- HCT116 cells were transfected with the indicated TOP3A plasmid constructs for 48 h. WT cells were co-transfected with either siControl or siCtIP for 72 h. Before harvest, cells were treated with either DMSO or the ATM inhibitor (ATMi) KU-55933 (20 μM) for 2 h. Protein-DNA adducts were isolated by RADAR assay and dot blotted with anti-TOP3A antibody. Representative slot blots are shown with quantitation of TOP3Accs to the right of each panel. Error bar represents data mean ± SD (n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett multiple comparison test. *Adjusted p-value = 0.0463 (Wild-type vs TDP2-/-), *Adjusted p-value = 0.0109 (Wild-type vs Wild-type+ATMi), ****Adjusted p-value = <0.0001 (TDP2-/- + ATMi) for (f) and **Adjusted p-value = 0.0079 (Wild-type vs siCtIP), **Adjusted p-value = 0.0035 (Wild-type vs Wild-type+ATMi), **Adjusted p-value = 0.0084 (Wild-type vs siCtIP + ATMi) for g. Source data are provided as a Source Data file.