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. 2016 Sep 16;7:12880. doi: 10.1038/ncomms12880

Figure 7. CDK-dependent phosphorylation of WRN at S1133 regulates DSB repair pathway choice at collapsed forks.

Figure 7

(a) Efficiency of HR-mediated repair of I-SceI-induced DSBs in HEK293TshWRN cells co-transfected with the indicated WRN forms, the I-SceI expression vector pCBASce and the pDRGFP HR reporter plasmid, as described in Methods. Western blotting shows expression of the indicated WRN forms in the HEK293TshWRN cells. The graphs show the percentage of HR efficiency calculated respect to cells transfected with the wild-type WRN protein. (b) End-joining efficiency of I-SceI-induced DSBs in HEK293TshWRN cells co-transfected with the indicated WRN forms, the pDRGFP NHEJ reporter plasmid linearized, as described in Methods. Western blotting shows expression of the indicated WRN forms in the HEK293TshWRN cells. The graph shows the percentage of NHEJ efficiency respect to cells transfected with the wild-type WRN. Data are presented as mean±s.e.m. from three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001; analysis of variance (ANOVA) test; n=3 × 105). (c) Analysis of sister chromatid exchanges. Cells were treated as indicated and recovered in BrdU-containing medium for 36 h before metaphase spreading and staining, as described in Supplementary Methods. The graph shows the mean number of sister chromatid exchanges (SCE) per metaphase cells. A minimum of 25 metaphases were counted for each experimental point from three independent experiments (NS, not significant; **P<0.01, ANOVA test; n=75). (d) Analysis of DSB repair efficiency. Cells were treated with 5 μM CPT for 1 h and allowed to recover for different time points as indicated. DSB repair was evaluated by the neutral Comet assay. In the graph, data are presented as mean tail moment±s.e.m. from three independent experiments (NS, not significant; ****P<0.0001; Mann–Whitney test; n=300). Representative images from the neutral Comet assay are shown in the panel. (e) Effect of DNA-PKcs inhibition on DSBs repair in the WRN phosphomutants. Cells were treated as indicated and allowed to recover in the presence or not of the DNA-PKcs inhibitor (DNA-PKi). The presence of DSBs was evaluated by the neutral Comet assay. In the graph, data are presented as mean tail moment±s.e.m. from three independent experiments (****P<0.0001; Mann–Whitney test; n=300).