SENP2 promotes DNA damage signaling and DNA repair. (A) IR colony survival in HeLa treated with siNTC or siSENP2 for 72 h. Cells were treated concurrently with 1 µg/mL doxycycline to induce siRNA-resistant forms of SENP2. n = 4. (B,C) HR (U2OS DR3-GFP) or NHEJ (U2OS-EJ5-GFP) assays using siSENP2 or siNTC treated cells transfected with RFP, I-SceI, and SENP2^WT or SENP2^C548A. GFP⁺ cells were normalized to RFP transfection efficiency. Percentage repair is given compared with siNTC. Western blot shows SENP2 knockdown efficiency and restoration with siRNA resistant cDNA. n = 3. (D,E) SUMO/γH2AX colocalizing foci in HeLa siNTC or siSENP2 cells fixed 1 h after 5 Gy of IR. (E) As for D with the indicated DDR factors. n = 3. (F–H) Time course of MDC1 (n = 200), GFP-RNF168 (n = 50), or 53BP1 (n = 150) foci in HeLa treated with indicated siRNA for 72 h. Representative images for 53BP1 foci at 4 h after IR are shown. (I–K) MDC1 and 53BP1 foci per cell, respectively, 4 h after 4 Gy of IR in siNTC or siSENP2 HeLa. (K) representative images related to I,J, n = 100 cells. (L,M) HeLa (siNTC/siSENP2) irradiated with 4 Gy and, 0.5 h later, treated with DMSO/0.1 µM VCPi, CB-5083. Cells were fixed at the indicated times and scored for MDC1 foci. (M) As for L but 53BP1 foci in cells fixed at 2 h. n = 100 cells.

MDC1 is a SENP2 substrate and hypo-SUMOylation of MDC1 permits DDR signaling. (A,B) HeLa treated with siRNA and induced with doxycycline for 72 h to express wild-type or K1840R myc-MDC1. Data show kinetics of foci per cell for the indicated times after treatment with 4 Gy of IR. (B) As for A but 53BP1 foci at 2 h. n = 100. (C). HEK293^FlpIn 6x-His-myc SUMO2 cells treated with the indicated siRNA (48 h), irradiated (10 Gy), lysed 1 h later, and subjected to Ni²⁺ agarose purification followed by immunoblotting with MDC1 antibodies to determine the relative enrichment in SUMO2 conjugates. (PD) Pull-downs. (D) Quantification of the MDC1 purified by Ni²⁺ pull-downs in HEK293^FlpIn 6xHis-myc-SUMO2 cells illustrated in C. Relative enrichment in SUMO2-MDC1 conjugates was determined by densitometry with the untreated siNTC sample being set as 1. n = 5. Error bars show SEM. (E) HEK293 transiently transfected with Flag-RFP-SENP2^WT for 48 h. Thirty minutes preirradiation (4 Gy), cells were treated with 10 µM ATMi or DMSO. Lysates were made at the indicated times and immunoprecipitated with Flag agarose and blotted for endogenous MDC1 and Flag-RFP-SENP2. (F) Quantification of relative Flag SENP2–MDC1 interaction ratio by densitometry, the ratio was set at 1 for untreated cells. n = 5. Error bars represent SEM. (G) HEK293^FlpIn myc-MDC1^WT transiently transfected with Flag-SENP2^WT or SENP2^ΔCC and treated with doxycycline for 72 h. Cells were irradiated (4 Gy) and lysed 1 h later followed by immunoprecipitation with myc-agarose. (H) HEK293^FlpIn 6x-His-myc SUMO2 cells, siRNA depleted for SENP2 and 24-h later transfected with SENP2^WT or SENP2^ΔCC for an additional 48 h. Cell lysis and Ni²⁺ pull-down was carried out as for C. (I,J). HeLa treated with siSENP2. Twenty-four hours later cells were transfected with Flag-SENP2 for 48 h, irradiated (4 Gy), and fixed at indicated times. (G) MDC1 foci per cell were measured in cells costaining with Flag-SENP2. n = 50. (K) Colony survival in IR (2 Gy) HeLa treated with siNTC, siSENP2, or siSENP2 plus doxycycline to induce expression of SENP2 mutants. n = 3.

RNF4-VCP is responsible for the IR sensitivity of SENP2-depleted cells. (A) IR colony survival in HeLa treated with the indicated siRNA for 72 h. (Right panel) Western blot of siRNA depletions. (B) Colony survival in HeLa^FlpIn RNF4 treated with the indicated siRNA and doxycycline to induce expression of RNF4 and its mutants for 72 h prior to 2 Gy IR, n = 3. Western blot is shown below. Note that RNF4 antibody will also detect exogenous protein. (C) Colony survival in HeLa^FlpIn SENP2 treated with indicated siRNA and doxycycline to induce expression of SENP2 and its mutants for 72 h prior to 2 Gy IR. n = 3. Western blot is shown below. (D) Colony survival of HeLa treated with the indicated siRNAs for 72 h and 4 Gy IR. Thirty minutes after irradiation, cells were treated with DMSO and 0.1 µM VCP inhibitor CB-5083 for an additional 90 min prior to plating for colonies in fresh media n = 3.

SENP2 is not relevant to S-phase clearance of MDC1. (A,B) HeLa treated with siNTC (black) or siSENP2 (blue) for 72 h, pulsed with 10 µM EdU 1 h prior to IR (4 Gy). Cells were fixed at indicated times and subjected to Click-It labeling with 647 nm azide to detect EdU incorporation into nascent chromatin. MDC1 foci per cell in EdU-negative (A) and EdU-positive (S phase) (B) cells. Fifty cells were scored per condition from a total of three experiments. Error bars represent SEM. (C) Representative images relating to A and B. (D) Western blot showing MDC1 knockdown and expression in HeLa^FlpIn myc-MDC1 wild type or K1840R (KR). (E–G) HeLa^FlpIn myc-MDC1^WT (blue) and K1840R (green) cells siRNA depleted for endogenous MDC1 and treated with doxycycline to induce MDC1. After 72 h, cells were treated with 2 Gy of IR (E), 10 µM olaparib (F), or 2.5 µM CPT (G) for 2 h and subjected to colony survival analysis. n = 3. (H) HeLa myc-MDC1^WT (blue) and K1840R (green) cells siRNA depleted for endogenous MDC1 and treated with doxycycline to induce myc-MDC1. After 72 h, cells were pulsed with 10 µM EdU 1 h prior to 4 Gy of IR. Cells were fixed 2 h after IR and subjected to Click-It labeling with 647-nm azide to detect EdU incorporation into nascent chromatin and stained with RAD51. n = 100 EdU⁺ cells. (I) HeLa treated with doxycycline to induce expression of myc-MDC1^WT or K1840R for 72 h. One hour prior to 4 Gy of IR, cells were pulsed with EdU and fixed 2 h later. Cells (100 from a total of three experiments) were scored for 53BP1 foci per cell in EdU^−/+ cells. (J,K). Colony survival in HeLa treated with indicated siRNA and 10 µM olaparib for 2 h (J) or 2.5 µM CPT for 2 h (K). n = 3. (L) U2OS-DR3 homologous recombination reporter cells treated with siRNA for 24 h prior to transfection with I-Sce-I nuclease and RFP (to control for transfection efficiency) for a further 48 h. The percentage of RFP/GFP-positive cells relative to siNTC is shown for three experiments. (M) HeLa depleted for indicated siRNA for 72 h were pulsed with 10 µM EdU for 1 h prior to 4 Gy of irradiation. Cells were fixed 4 h later and RAD51 foci counted in EdU-positive cells. n = 100. (N) Images relating to M.

HR is sensitive to the supply of SUMO. (A) Colony survival after 2 Gy IR in HeLa^FlpIn 6xHis-myc SUMO in siNTC/siSENP2-depleted cells. GA indicates diglycine → alanine mutants in SUMO isoforms that prevent conjugation. n = 3. (B) HeLa treated with siRNA for 24 h before transfection with myc-SUMO; 48 h later cells were treated with 4 Gy, fixed at 4 h, immunostained, and scored for MDC1 in myc-SUMO expressing cells. n = 100. (C) Western blot of SUMO conjugates relating to (A,B). (D,E) As for A but using 1 µM CPT (D) or 10 µM olaparib (E) for 2 h before plating for colony survival. n = 3. (F). HeLa treated with siRNA for 24 h before transfection with myc-SUMO for 48 h. Cells were incubated with 10 µM EdU 30 min prior to 4 Gy of IR, fixed at 4 h, and immunostained for RAD51 and myc to detect myc-SUMO-expressing cells. n = 100. Only EdU⁺ myc-SUMO-positive cells were counted for RAD51 focus number. (G) Western blot showing partial depletion of SUMO2/3 conjugates in HeLa. (H) Colony survival in HeLa depleted with siNTC or siSUMO2/3 followed by treatment with 2 Gy of IR, 1 µM CPT, or 10 µM olaparib. (I). Western blot showing SUMO2/3 knockdown. (J) U2OS HR and NHEJ reporters treated with siNTC or siSUMO2/3 and transfected with i-Sce-I and RFP for 72 h. HR and NHEJ efficiency was set at 100% for siNTC. (K) HAP1 cells synchronized into G1-, S-, and M-enriched populations; lysed; and probed for free SUMO2/3. Cyclin A1 is a late S-phase marker.

SENP2 overexpression disrupts responses to DSBs. (A,B) HeLa^FlpIn SENP2^WT mock or doxycycline (to induce SENP2 expression) for 72 h were treated with indicated dose of IR and subjected to colony survival analysis. (B) Colony assay performed as for A but with siRNA transfection concurrent with doxycycline addition. IR = 2 Gy. n = 3. (C,D) HeLa transfected with SENP2^WT for 48 h prior to 4 Gy IR and fixation 2 h later. MDC1 foci per cell were scored in Flag-SENP2-positive cells. n = 100. (D) Representative images of C. (E,F) HR and NHEJ U2OS reporters expressing RFP or RFP-SENP2 and I-Sce1 GFP-positive cells were normalized to RFP-transfection efficiency. Percentage repair is given compared with NTC. (F) Western blot showing expression of RFP-SENP2. n = 4. (G) HeLa transfected with SENP2^WT for 48 h prior to 30 min of EdU pulse and 4 Gy of IR followed by fixation 4 h later. One-hundred EdU/Flag-SENP2-positive and 100 EdU/untransfected cells were scored for RAD51 foci across three experiments. (H) As for A but using 2 h of treatment of 2.5 µM CPT for 2 h prior to plating. (I) HeLa SENP2^WT or HeLa treated with doxycycline for 72 h prior to IR 2 Gy. Eighteen hours later, cells were treated with colcimid for 6 h and processed for metaphase spread analysis. Data show percentage metaphases with chromosome/chromatid gaps from three experiments. Error bars represent SEM.