Fig. 7.

DDX11 facilitates resection and RPA loading to ssDNA substrates. (A) Quantification and representative micrographs of RPA32 foci in U2OS Ctrl and DDX11 KO cells recovering from an acute treatment with cisplatin (2.5 μM for 1 h) after 24 h. (Scale bar, 10 μm.) n = 2. Statistical analysis of foci was performed using Student’s t test. Error bars show average ± SD. (B) U2OS DIvA cells were transfected with siCtrl and siDDX11 followed by the addition of 4OHT (300 nM) to induce AsiSI-mediated DSBs after 48 h of post-transfection. Resection assay at the two different regions (KDELR3 and ASXL1) were analyzed after 4 h of 4OHT by real-time PCR. Values were normalized against the amount of ssDNA detected in control cells prior to 4OHT treatment, n ≥ 3. Corresponding Western blot and immunofluorescence for AsiSI-induced DSBs is shown. (Scale bar, 10 μm.) Statistical analysis was performed using Student’s t test. Error bars show average ± SEM. (C) Model for the DDX11 proposed role in resolving secondary structures upon DSB end resection to facilitate RPA loading and subsequently RAD51 nucleofilament formation required for homologous recombination-mediated DSB repair (see text for details).