Fig 2: Antagonizing USP1 by APCCdh1 at DNA damage sites is critical for BRCA1 recruitment.(a,b). Representative images of BRCA1 (a) and RIF1 (b) IRIF in U2OS-Fucci cells transfected with the indicated siRNAs and expression vectors. The cells were irradiated with 2 Gy and recovered for 1 h before the immunostaining. (c,d) Quantification of BRCA1 (c, n>226) and RIF1 (d, n>195) focus formation in a,b. Blue box designates cells with more than 20 (BRCA1 positive) and 10 (RIF1 positive) foci, respectively. The percentages of BRCA1-positive and RIF1-positive cells are indicated above each box. Scale bars represent 10 μm.

Fig 3: RIF1 and BRCA1 IRIF co-exist in S/G2 cells.(a) Co-staining of BRCA1 and RIF1 in U2OS cells 1 h after 2 Gy IR. (b) Quantification of RIF1 focus number per cell in U2OS-Fucci cells transfected with control or two different BRCA1 siRNAs. The cells were subjected to 2 Gy IR 3 days after the siRNA transfection. Left panel: representative images of RIF1 foci. Right panel: quantification of RIF1 focus number in the cells (n>212). (c) Co-staining of BRCA1 and 53BP1 in U2OS cells 1 h after 2 Gy IR. (d) Quantification of BRCA1 focus per cell in U2OS-Fucci cells transfected with control or two different RIF1 siRNAs. Cells were subjected to 2 Gy IR 3 days after the siRNA transfection. Left panel: representative images of BRCA1 foci. Right panel: quantification of BRCA1 focus number in the cells (n>188). The intensity profile graphs show the immunofluorescence intensities of the indicated proteins along the line on the images (a,c). Red lines on the graphs indicate the mean number of RIF1 (b) or BRCA1 (d) foci per cell. Blue boxes designate cells with more than 10 RIF1 foci (b) or 15 BRCA1 foci (d). The percentages of RIF1-positive (>10 foci) or BRCA1-positive (>15 foci) cells are indicated above each blue box. ** (in b) indicates P<0.001 (student’s t-test). Scale bars represent 10 μm.

Fig 4: A model for the regulation of DSB repair pathway choice by USP1 and APCCdh1 in S/G2 cells.The DSBs in S/G2 cells can be processed by two competing factors, the Ku heterodimers and the MRN complex. (a) When a DSB is bound by Ku heterodimers, the break is then destined for NHEJ. (b) On the other hand, when a site is bound by MRN complex, the ends are resected and ssDNA generated, leading to the activation of ATR, Chk1 and APCCdh1, and eventually the destruction of USP1 and the recruitment of BRCA1. BRCA1 then expels RIF1 and reinforces the end resection, leading to further activation of ATR, Chk1 and APC.

Fig 5: USP1 is targeted by APCCdh1 at DSBs.(a) The K11-linked ubiquitin chains were primarily formed on USP1. U2OS cells transfected with control or USP1 siRNA were microirradiated and processed for immunostaining 30 min later. (b) The mobilization kinetics of GFP-USP1 to sites of DNA damage. GFP-USP1 was expressed in U2OS cells transfected with the indicated siRNAs and its mobilization was monitored for 2 h following the microirradiation. (c) In vivo ubiquitination assay of endogenous USP1 in the presence or absence of Cdh1 after DNA damage. Cell lysates were prepared as described in methods. (d) Quantification of BRCA1 focus number and intensity in Cdh1- and APC3-depleted cells. The cells were subjected to 2 Gy IR 3 days after the siRNA transfection. Top panel: quantification of BRCA1 foci per cell. Blue box designates the cells with more than 15 foci, whose percentage is indicated above each box. Bottom panel: average intensities of BRCA1 focus per cell (n>195). (e) The accumulation of endogenous K63-linked ubiquitin chains at damage sites. U2OS cells were microirradiated and recovered for 30 min before the immunofluorescence analysis of K63-linked Ub and γH2AX. Left panel: representative images of K63-linked Ub and γH2AX. Right panel: quantification of K63-linked Ub at microirradiated regions (n>102). (f) Quantification of BRCA1 (n>191) and RIF1 (n>197) focus formation in U2OS-Fucci cells transfected with the indicated siRNAs and siCdh1-resistant Cdh1 constructs. Blue box designates the cells with more than 20 (BRCA1 positive) and 10 (RIF1 positive) foci, respectively. The percentages of BRCA1-positive and RIF1-positive cells are indicated above each box. (g) Quantification of BRCA1 focus number in the cells transfected with the indicated siRNAs (n>200). (h) In vivo ubiquitination of H2AX in Cdh1-, USP1- or both Cdh1- and USP1-depleted cells. The indicated siRNAs were transfected first, followed by GFP-RNF168 transfection 48 h later, and another 24 h later, the cells were irradiated with 10 Gy and harvested 1 h after IR for the analysis of H2AX ubiquitination. Error bars indicate SEM from three independent experiments. ** indicates P<0.001 (student’s t-test). Scale bars represent 10 μm.