Fig 2: Recombinant Human TDP2 Excises Denatured but Not Native TOP3Bcc(A) Oligonucleotide substrate (69-mer) with main TOP3B site (17 nucleotides from the 5' end).(B) Recombinant human TDP2 does not excise native TOP3Bccs. The oligonucleotide (300 nM) was incubated with recombinant human TOP3B (4 µM). TOP3Bcc formation results in a slower migrating band (lane 2) (for full gel and additional minor bands see Figure S3A). TOP3Bccs were incubated with 1 or 3 µM recombinant TDP1 (lanes 7 and 8) or TDP2 (lanes 5 and 6). Benzonase (3 and 9 units, lanes 3 and 4,) was used as a positive control to degrade the oligonucleotide and release TOP3B. Samples were immunoblotted with anti-TOP3B antibody after SDS-PAGE.(C) Recombinant human TDP2 excises denatured cellular DNA and RNA TOP3Bccs. HEK293 cells were transfected with FLAG-tagged R338W-TOP3B, and protein-nucleic acid adducts were recovered by RADAR assay. After incubation with recombinant TDP2 (3 and 6 µM, lanes 4 and 5), H351A TDP2 (3 and 6 µM, lanes 6 and 7), D262A TDP2 (3 and 6 µM, lanes 8 and 9), benzonase (250 units, lane 2), or MNase (300 units, lane 3), released TOP3B was detected by immunoblotting with anti-FLAG antibody after SDS-PAGE. Loading was tested by slot blotting and probing with anti-dsDNA antibody.(D) Recombinant human TDP2 excises cellular RNA TOP3Bccs. HEK293 cells were transfected with R338W-TOP3B. Covalent protein-RNA adducts were isolated using TRIzol. After incubation with recombinant TDP2 (3 and 6 µM, lanes 4 and 5), H351A TDP2 (3 and 6 µM, lanes 6 and 7), D262A TDP2 (3 and 6 µM, lanes 8 and 9), benzonase (250 units, lane 2), or an excess amount of RNase A (200 µg/mL) and RNase T1 (200 units/ml) mix (lane 3), released TOP3B was detected by immunoblotting with anti-FLAG antibody after SDS-PAGE. Loading was tested by methylene blue staining (RNA).See also Figure S3.

Fig 3: Analysis of DPC Repair Deficiency in SPRTN and TDP1/TDP2 Knockout Mutants with the ARK Assay(A) Removal of DPCs in wild-type TK6 cells and 2 SPRTN knockout derivatives (KO1 and KO2). Cells were exposed to 400 μM FA for 2 h. DPC coefficients were determined for each cell line at the indicated time points and normalized against the 0 time point to arrive at the percentages of DPCs remaining.(B) DPC accumulation in wild-type TK6 cells and 2 SPRTN knockout derivatives continuously exposed to low-dose FA (50 μM) for 12 and 24 h.(C) DPC accumulation in wild-type TK6 cells and a TDP1/2−/− double-knockout derivative continuously exposed to CPT (75 nM) for 12 and 24 h.Each data point in the plots and in the bar graphs was derived from no less than 5 biological repeats with duplicates or triplicates. One-way ANOVA analyses for the three time points 6, 9, and 24 h generated p < 0.0001 in (A) for the F-test and interested pairwise Tukey test results are indicated. Number of biological repeats with triplication = 3. The error bars depict standard deviations.

Fig 4: TDP2-Mediated Repair of TOP3Bccs Is Dependent on Ubiquitination and Proteasomal Processing(A) WT and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W-TOP3B alone and incubated for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h) as indicated. Nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay. TOP3Bccs were detected using anti-FLAG antibody. Equal loading was with anti-dsDNA antibody. The figure is representative of two independent experiments.(B) Quantitation of TOP3Bccs in two independent experiments as shown in (A).(C) WT and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W-TOP3B alone and incubated for 72 h. Before harvest, cells were treated with TAK243 (10 µM, 2 h). Nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay, and TOP3Bccs were detected using anti-FLAG antibody. Equal loading was tested with anti-dsDNA antibody. The figure is representative of two independent experiments.(D) Quantitation of TOP3Bccs in two independent RADAR assays as shown in (C).(E) WT and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W-TOP3B alone or co-transfected with siTRIM41 constructs and incubated for 72 h. TOP3Bccs were detected after RADAR assay with anti-FLAG antibody. Equal loading was tested with anti-dsDNA antibody. The figure is representative of two independent experiments.(F) Quantitation of TOP3Bccs in two independent RADAR assays as shown in (E).(G) Model for the processing of TOP3Bccs by TRIM14, the proteasome (PSM), and TDP2. See also Figure S6.

Fig 5: TDP2 Excises Cellular TOP3Bccs from Both DNA and RNA(A) WT or TDP2KO HCT116 cells were transfected with R338W-TOP3B. After 72 h, protein-nucleic acid adducts were isolated by RADAR assay. Slot-blotted TOP3Bccs were detected with anti-FLAG antibody. Loading was tested by slot blotting and probing with anti-dsDNA antibody.(B) Quantitation of TOP3Bccs from three independent experiments as shown in (A). TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration.(C) HCT116 WT and TDP2KO cells were transfected with WT-TOP3B or R338W-TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to isolate DNA and RNA fractions. TOP3Bccs were detected with anti-FLAG antibody. Loading was tested with anti-dsDNA antibody or methylene blue staining (RNA).(D) Quantitation of TOP3Bcc in three independent experiments as shown in (C).(E) WT and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W-TOP3B. After 72 h, protein-RNA adducts were isolated using TRIzol. TOP3Bccs were detected with anti-FLAG antibody. Loading was tested by methylene blue staining.(F) Quantitation of TOP3Bcc in RNA in three independent experiments as shown in (E).(G) Ectopic expression of TDP2 reduces TOP3Bccs. WT and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W-TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, TOP3Bccs were detected with anti-FLAG antibody. Loading was tested with anti-dsDNA antibody.(H) Quantitation of TOP3Bccs in three independent experiments as shown in (G).See also Figure S2.