Fig 2: USP52 regulates DNA end resection and HR through CtIP.a ER-AsiSI U2OS cells with or without USP52 depletion were transfected with indicated constructs for 24 h, then cells were treated with 1 μM 4-OHT for 4 h before genomic DNA was extracted and digested with BsrGI. DNA end resection efficiency was measured by qPCR assay. Data are presented as mean values ± SEM from three independent experiments. b–e Control or USP52-depleted U2OS cells were transfected with indicated constructs for 24 h before treated with 2 Gy IR for 2 h (b, c) or 5 Gy IR for 5 h (d, e). RPA2 or Rad51 focus formation was then detected by immunofluorescence (b, d) and quantified (c, e). Data are representative of three independent experiments. Each dot represents a single cell, and more than 200 cells were counted in each group for this experiment. Error bars represent SEM from this experiment. Scale bar, 10 μm. f Control or USP52-depleted HEK293T cells reconstituted with the indicated constructs and HR reporter were subjected to HR assay. Error bars represent SEM from three independent experiments. g Control or USP52-depleted ER-AsiSI U2OS cells were transfected with control or CtIP shRNA, and then cells were treated with 4-OHT for DNA end resection assay. Each bar represents SEM from three independent experiments. h Control or USP52-depleted HEK293T cells were transfected with control or CtIP shRNA and HR reporter, and then cells were harvested for HR assay. Error bars represent SEM from three independent experiments. i Control or USP52-depleted HEK293T cells were transfected with WT USP52 or USP52 ΔUCH for 24 h before treated with 5 Gy IR for 1 h. Cells were harvested and subject to immunoprecipitation with control IgG and CtIP antibodies. The immunoprecipitates were then blotted with indicated antibodies. j, k Control and USP52-depleted U2OS cells stably expressing WT USP52 or ∆UCH were in response to the indicated dose of IR (j) or PARPi (k) for 2 weeks. Cell viability was assessed using colony formation assay. Error bars represent SEM from three independent experiments.

Fig 3: ATM kinase promotes the activity of USP52 to regulate the DDR.a HEK293T cells transfected with FLAG–USP52 were treated with DMSO or 25 µM Ku55933 for 2 h prior to IR treatment. Harvested cells were immunoprecipitated with anti-FLAG agarose beads. After untreated or treated with lambda protein phosphatase, blots were probed with pSQ/TQ antibody. b HEK293T cells transfected with indicated USP52 constructs were harvested after IR and then immunoprecipitated with anti-FLAG agarose, blots were probed with indicated antibodies. c Ubiquitinated CtIP was incubated with purified FLAG-WT USP52 or the S1003A mutant before or after IR to perform deubiquitination reaction assay in vitro, and then blotted with the indicated antibodies. d, g Control or USP52-depleted U2OS cells were transfected with indicated USP52 truncates before treated with 2 Gy IR for 2 h or 5 Gy IR for 5 h. RPA2 and Rad51 focus formation were detected by immunofluorescence (d, f) and quantified (e, g). Data are representative of three independent experiments. Each dot represents a single cell, and more than 200 cells were counted in each group for this experiment. Error bars represent SEM from this experiment. Scale bar, 10 µm. h Control or USP52-depleted HEK293T cells transfected with indicated USP52 constructs together with HR reporter were harvested for HR assay. Error bars represent SEM from three independent experiments. i USP52-depleted HEK293T cells were transfected with indicated USP52 constructs for 24 h before treated with or without IR. Harvested cells were then immunoprecipitated with nickel (His) beads and blotted with indicated antibodies. j Purified WT USP52 or the S1003A mutant was incubated with ATM for in vitro kinase assay, and then ATM-phosphorylated USP52 constructs were used for in vitro deubiquitination reaction with ubiquitinated GFP-CtIP. k Control or USP52-depleted HEK293T cells transfected with indicated USP52 constructs were harvested and immunoprecipitated with anti-FLAG agarose, and then subjected to blot with the indicated antibodies. l, m Control or USP52-depleted U2OS cells stably expressing indicated USP52 constructs were treated with IR or PARPi for 2 weeks. Cell viability was assessed using colony formation assay. Error bars represent SEM from three independent experiments.

Fig 4: USP52 promotes DNA end resection and HR repair.a, b, f–i Representative pictures (a, f, h) and quantifications (b, g, i) of CtIP (a, b), RPA2 (f, g) and Rad51 (h, i) foci in control or USP52-depleted U2OS cells when treated with 5 Gy IR for 3 h, 2 Gy IR for 2 h or 5 Gy IR for 5 h, respectively. Data are representative of three independent experiments. Each dot represents a single cell, and more than 200 cells were counted in each group for this experiment. Error bars represent SEM from this experiment. Scale bar, 10 µm. c Illustration of the designation of Taqman qPCR primers and probes (black arrows) for measuring resection at sites adjacent to the AsiSI sites (red arrows). The primer pairs are across BsrGI restriction sites. All Taqman probes are designed at either side of the restriction site. d Control or USP52-depleted ER-AsiSI U2OS cells transfected with FLAG–CtIP were added with or without 4-OHT. ChIP assay was then performed using FLAG antibody. e The genomic DNA extracted from control or USP52-depleted ER-AsiSI U2OS cells were digested with BsrGI. DNA end resection adjacent to DNA double-strand break sites was measured by qPCR assay. j The HR-mediated DSB repair efficacy of control and USP52 depletion in HEK293T cells were analyzed using HR reporter. Data are presented as mean values ± SEM from three independent experiments. k Control or USP52-depleted ER-AsiSI U2OS cells transfected with indicated constructs were used for DNA end resection assay. Data are presented as mean values ± SEM from four independent experiments. l The efficacy of HR in control or USP52-depleted HEK293T cells transfected with indicated USP52 constructs were analyzed using GFP reporter assay. m, n Control or USP52-depleted U2OS cells were exposed to the indicated dose of IR (m) or PARPi (n) for 2 weeks, colony formation assay was then performed to detect the survival rate of cells. d, e, j, l–n Data are presented as mean values ± SEM from three independent experiments.

Fig 5: Deubiquitination of CtIP by USP52 is important for DNA end resection and HR.a HEK293T cells were transfected with WT CtIP or CtIP 2KR for 24 h before harvesting and immunoprecipitation with nickel (His) beads. Blots were detected by indicated antibodies. b Control or USP52-depleted ER-AsiSI U2OS cells were transfected with WT CtIP or CtIP 2KR for 24 h, and then cells were treated with 4-OHT to induce DSB. Cells were harvested for DNA end resection analysis measured by qPCR assay. Each bar represents SEM from three independent experiments. c–f Representative images (c, e) and quantification (d, f) of RPA2 (c, d) and Rad51 foci (e, f) in control or USP52 knockdown U2OS cells which were transfected with WT CtIP or CtIP 2KR for 24 h before treated with 2 Gy IR for another 2 h or 5 Gy IR for 5 h. Data are representative of three independent experiments. Each dot represents a single cell, and more than 200 cells were counted in each group for this experiment. Error bars represent SEM from this experiment. Scale bar, 10 µm. g Control or USP52-depleted HEK293T cells which were transfected with WT CtIP or CtIP 2KR were subjected to DR-GFP-based HR assay. Data are presented as mean values ± SEM from three independent experiments. h Control or USP52-depleted HEK293T cells were transfected with WT or 2KR mutant of CtIP for 24 h prior to be treated with 5 Gy IR. Cells were then harvested and immunoprecipitated with anti-FLAG agarose beads to detect the phosphorylation of CtIP at T847. i, j The sensitivity of control or USP52-depleted cells stably expressing WT CtIP or 2KR truncates in response to IR (i) or PARPi (j) were analyzed by colony formation assay. Error bars represent SEM from three independent experiments.