Figure 6. ATM-dependent phosphorylation of Rad17 at Thr622 is required for HR repair.

A Schematic presentation of potential phosphorylation TQ/SQ motifs in Rad17.

B ATM directly phosphorylates Rad17 at Thr622. An in vitro ATM kinase assay was performed as described in Supplementary Material and Methods.

C U2OS cells were either untreated or exposed to 5 Gy of IR. Western blot was performed as indicated.

D ATM but not ATR is responsible for Rad17 phosphorylation at Thr622 following DSB damage. U2OS cells transfected with the indicated siRNA were either untreated or exposed to 5 Gy of IR. Cell lysates were prepared 1 h after IR. Western blot was performed.

E Endogenous Rad17 is depleted by Dox induction and replaced by exogenous Rad17. Inducible Rad17 knockdown cells (U2OS) stably expressing shRNA-resistant Rad17 WT or Rad17 T622A mutant were treated with or without Dox for 2 days. Western blot was performed.

F–H Rad17 Thr622 phosphorylation is required for HR repair and cell survival. Inducible Rad17-knockdown cells reconstituted with shRNA-resistant Rad17 WT or Rad17 T622A mutant were used. HR repair assay was performed as previously described (F); comet assay was performed as previously described (G); clonogenic survival assay was performed using IR treatment (H).

I, J Rad17 Thr622 phosphorylation is necessary for tumor growth in the presence of PARP inhibitor. Mice bearing xenograft tumors of 200–220 mm³ were divided into two groups for each cell line as indicated: vehicle control group and Olaparib group. Each group contained four nude mice. Tumor size was measured and tumor growth curves plotted (I); tumors were harvested on day 28 after the start of Olaparib treatment (J).

Data information: Error bars represent mean ± s.d. (n = 4).

Source data are available online for this figure.