Fig. 4: PARP1 orchestrates TOP1cc and TOP1cc-like DPC repair.

A Schematic illustrating the current model of TOP1cc repair. B PARP1 deletion suppresses the visibility of camptothecin-induced SSBs. The indicated RPE-1 cells were incubated with 10 µM camptothecin for 1 h and then processed for alkaline comet assays to measure unrepaired SSBs. Data show the median Tail moment of 300 cells combined from three independent experiments (100 cells/experiment). Significant differences were determined by 2-way ANOVA with Sidak’s post hoc multiple comparisons test. CPT denotes camptothecin. **** denotes p-value ≤0.0001. C Proteasome inhibition and/or PARP1 deletion suppresses the visibility of camptothecin-induced SSBs in the alkaline comet assay pre-treated with proteasome inhibitor (50 µM MG132) 2 h prior to and during camptothecin treatment as above. D To generate pFLP, Flp-nick is crosslinked to a plasmid containing the FRT site. Products of Flp-nick incubation with a CTRL (pCTRL) or FRT-site containing plasmid (pFRT) were analyzed on a native agarose gel. E pFLP was incubated in non-replicating egg extracts (1:2 HSS:NPE ratio) in the absence and presence of Ub.E1i. Reaction samples were analyzed by native agarose gel electrophoresis. F Quantification of the experiment shown in E. Error bars represents the SD of the mean. n = 3 independent experiments. Significant differences were determined by a two-tailed unpaired t-test. ** denotes p-value ≤0.01 (p = 0.0061). G Analysis of protein recruitment to pFLP compared to pCTRL via PP-MS. Plasmids were recovered at 10 min after addition in non-replicating egg extracts (1:2 HSS:NPE ratio). The volcano plot shows the difference in the abundance of proteins between the two sample conditions (x-axis), plotted against the p-value resulting from two-tailed Student’s two-sample t-testing (y-axis). Proteins significantly down- or up-regulated (cutoff line represents permutation-based FDR < 5%) are represented in red or blue, respectively. n = 4. H pFLP was incubated in non-replicating egg extracts (1:2 HSS:NPE ratio) in the absence and presence of the indicated inhibitors. Reaction samples were analyzed as in (E). I Quantification of the experiment shown in (H) as in (F). Error bars represent the SD of the mean. n = 3 independent experiments. Significant differences were determined by a two-tailed unpaired t-test. ** denotes p-value ≤0.01 compared to the Mock control (p = 0.0052 for PARPi and p = 0.0061 for Ub.E1i). J Add-back rescue experiment with WT and E988K PARP1 in non-replicating egg extracts (1:2 HSS:NPE ratio). Reaction samples were analyzed as in (E). K Samples from (J) were quantified as in (F). Error bars represent the SD of the mean. n = 3 independent experiments. Significant differences were determined by a two-tailed unpaired t-test. *** and **** denote p-value ≤0.001 and p-value ≤ 0.0001 compared to the Mock control, respectively. ** denotes p-value ≤0.01 compared to the PARP1 depletion control (p = 0.0054). * denotes p-value ≤0.05 compared to Mock control (p = 0.02). L pFLP^PK was incubated in non-replicating egg extracts (1:2 HSS:NPE ratio) egg extracts in the absence and presence of Ub.E1i or in PARP1-depleted egg extracts. Reaction samples were as in E. Source data are provided as a Source Data file.