Fig. 4: DePARylation of TOP1-DPCs is required for their proteasomal degradation.

a. Scheme for biotinylated TOP1cc preparation, PARylation, and proteasome digestion assays. b TOP1cc proteasomal digestion assay showing that unmodified TOP1ccs (without NAD⁺) are fully degraded by the 26S proteasome and partially degraded by the 20S proteasome after 45 min incubation whereas PARylated TOP1ccs (+ NAD⁺) are refractory to proteasomal degradation. c Inhibiting PARG blocked the interactions between TOP1-DPC and the proteasome subunit PSMD14. Following transfection of PSMD14-FLAG expression plasmid, HEK293 cells were treated with the indicated drugs (1 h, 10 µM PARGi pre-treatment followed by 30 min co-treatment with 20 µM CPT) and subjected to IP using an α-FLAG antibody. The immunoprecipitates and input samples were Western blotted with the indicated antibodies. d Inhibiting PARG blocked the interactions between TOP1-DPC and the proteasome subunit PSMB5. Following transfection of PSMB5-FLAG expression plasmid, HEK293 cells were treated with the indicated drugs (1 h, 10 µM PARGi pre-treatment followed by 30 min co-treatment with 20 µM CPT) and subjected to IP using an α-FLAG antibody. The immunoprecipitates and input samples were Western blotted with the indicated antibodies. e. Model for 26 S proteasome-dependent degradation of TOP1-DPCs. f Inhibiting PARG blocked the liberation of TOP1-induced DNA breaks. Upper panels: representative images of alkaline comet assay in HEK293 cells treated with DMSO, CPT (10 µM, 2 h), PARGi (10 µM, 3 h), and CPT + PARGi (pre-treatment with PARGi for 1 h then co-treatment with CPT and PARGi for 2 h). Cells were subjected to alkaline comet assay for detection of DNA breaks. Lower panel: quantitation of tail moments using OpenComet. n = 50 biologically independent cells. P value was calculated by paired Student’s t-test (two-tailed distribution). The scale bar represents 100 μm. g Inhibiting PARG attenuated TOP1-DPC-induced DNA damage response (DDR). HEK293 cells were treated with CPT in absence of the presence of PARGi (pre-treatment for 1 h). Cells were collected at the indicated time points and subjected to non-denaturing lysis and benzonase treatment, followed by Western blotting with the indicated antibodies. h Inhibiting PARG reduced CPT-induced γH2AX foci. Upper panels: representative images of IF of γH2AX and IdU foci by iSIM. U2OS cells were synchronized in the S phase by double thymidine block, followed by IdU incorporation and PARGi treatment for 1 h. Cells were then treated with CPT (1 µM) and collected at indicated time points for IF using anti-γH2AX and anti-BrdU antibodies. Lower panels: quantitation of γH2AX foci (left) and quantitation of IdU foci (right). n = 10 biologically independent samples. Data are presented as mean values +/− standard deviation (SD). P value was calculated by paired Student’s t-test (two-tailed distribution). ***: p = 0.000015. NS not significant. The scale bar represents 10 μm.