Fig. 5

Senataxin depletion decreases HR but does not impede resection. a Rad51 staining performed in untreated or 4OHT-treated DIvA cells (4 h), after transfection with control or SETX siRNA as indicated. Scale bar: 10 µM. Right panel shows the quantification of the Rad51 nuclear signal within foci (>100 nuclei) from a representative experiment. Center line: median; box limits: 1st and 3rd quartiles; whiskers: maximum and minimum without outliers. P values are indicated (unpaired t-test). b 53BP1 staining performed in untreated or 4OHT-treated DIvA cells (4 h), after transfection with control or SETX siRNA as indicated. Scale bar: 10 µM. Right panel shows the quantification of the 53BP1 nuclear signal within foci ( > 100 nuclei) from a representative experiment. Center line: median; box limits: 1st and 3rd quartiles; whiskers: maximum and minimum without outliers. P values are indicated (unpaired t-test). c HR (top panel), SSA (middle panel), and NHEJ (bottom panel) usage was measured using cell lines harboring specific reporter constructs^38,39,40 in control or senataxin-deficient (siRNA-transfected) dedicated cells. Myc-I-SceI expression was controlled by western blot in each condition. Mean and s.e.m. of at least three biological replicates are shown (as indicated). P values are indicated (paired t-test). d The site specific resection assay has been described earlier. Briefly, DNA purified from damaged or undamaged cells is digested by dedicated restriction enzymes (as indicated) and digestion-resistant DNA (single stranded DNA) is measured by qPCR, using primers pairs apart from the restriction sites. Here, we optimized this assay at two AsiSI-induced DSBs that were shown to undergo HR²⁹. e Resection assay at the two DSBs in control or SETX-siRNA transfected cells. Values were normalized against the % of ssDNA detected in control cells before 4OHT treatment. Average and s.e.m. of four biological replicates are shown. P values are indicated (paired t-test)