Figure 1.

Loss of WRN exonuclease activity leads to formation of nascent ssDNA which compromises formation of DSBs in response to a low-dose of camptothecin. (A) Evaluation of ssDNA by anti-IdU immunofluorescence under non-denaturing condition. Nascent DNA was pre-labeled for 15 min with IdU before treatment and labeling remained during treatment with CPT. Dot plots show the mean intensity of ssDNA staining for single nuclei from cells expressing the wild-type (WS^WT) or the exo-dead form of WRN (WS^E84A). Cells were either left untreated or challenged with 50 nM CPT for increasing periods, as indicated. The intensity of the anti-IdU immunofluorescence was measured in at least 200 nuclei from three independent experiments. Values are represented as means ±SE. Representative images of ssDNA labeling are shown. (B) Evaluation of nascent ssDNA in cells treated with nuclease inhibitors. Cells were treated with Mirin, C5 or both 30 min before IdU labeling and 45 min before CPT treatment for 4 h, and then subjected to the ssDNA assay. The graph shows the mean intensity of IdU fluorescence measured from two independent experiments (n = 200), data are presented as mean ± SE. Statistical analysis in A and B was performed by the Mann–Whitney test (**P < 0.01; ***P < 0.001; ****P < 0.0001). (C) Analysis of DSB accumulation by the neutral Comet assay. Cells were treated or not with CPT 50 nM for the indicated time, or with 5μM CPT (high-dose) for 1 h, and then subjected to the neutral Comet assay. Where indicated, cells were pre-treated with Mirin, C5 or both. In the graph, data are presented as mean tail moment ± SE from two independent experiments (ns = not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001; ANOVA test). Representative images from the neutral Comet assay are shown.