Fig. 3: WRN helicase inhibition triggers SNF2 translocase-dependent nascent DNA degradation by MRE11 nuclease in BRCA2-mutated cancer cells.

a Fork stability assay in PEO1 and PEO4 cells exposed to WRNi NSC617145. Cells transfected with either control or WRN siRNA (80 nM) were sequentially labeled with CldU and IdU and subjected to WRNi (NSC617145) treatment (4 µM) for 5 h. For untreated control, cells were collected immediately after labeling. Scatter plot showing IdU/CldU tract length ratios in individual experimental conditions. Representative of n = 2 independent experiments; p-values (p < 0.0001, p < 0.0001) were derived from n ≥ 150 DNA fibers using two-tailed Mann–Whitney test. Immunoblots showing WRN knockdown. b Fork stability assay in NSC617145-treated PEO1 and PEO4 cells upon Mirin (left panel) or DNA2i (right panel) treatment. Representative fibers of the Mirin experiment are shown. Representative of n = 2 independent experiments; p-values (p = 0.0542, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.6094) were derived from n ≥ 125 DNA fibers using two-tailed Mann–Whitney test. c Immunoblots showing dose-dependent chromatin enrichment of MRE11 upon WRNi treatment. Cells were treated with indicated doses of WRNi (NSC617145) for 1 h and subjected to subcellular fractionation. EXO1 level is shown. ORC2 was used as a positive marker and loading control for chromatin fractions. d Fork stability assay in PEO1 cells depleted of SMARCAL1, ZRANB3, or HLTF upon NSC617145 treatment. (Left panel) Immunoblots showing knockdown. Representative DNA fibers are shown. (Right panel) Quantification of IdU/CldU ratios in individual experimental conditions. Representative of n = 2 independent experiments; p-values (p < 0.0001, p < 0.0001, p < 0.0001) were derived from n ≥ 250 DNA fibers using two-tailed Mann–Whitney test. In a, b, and d horizontal red bars indicate the median of IdU/CldU ratios; median IdU/CldU values are indicated; purple and green colors indicate CldU and IdU labeling, respectively. β-actin was used as a loading control in immunoblots in (a) and (d). Western blots a, c, d were repeated independently at least two times with similar results. Source data are provided as a Source Data file.