ATP-dependent assembly of a DNA damage signaling complex. (A) Nuclear extracts were incubated for 10 min at 20°C with gDNA with or without 1 mM AMP-PNP or 1 mM ATP as indicated. Next, the reaction mixture was incubated overnight with an anti-ATR antibody, and ATR-associated proteins were pulled down with protein G–coupled magnetic beads, resolved by SDS-PAGE, and revealed by Western blotting with the indicated antibodies. (B) ATR immunoprecipitations were conducted as described in A and probed for ATM by Western blotting. (C) Reactions mixtures were assembled as described in A in the presence of 1 mM ATP, with or without 100 µM IC86621, as indicated. (D) Model for the concerted activation of DNA-PKcs and ATR. RPA binds to the ssDNA gap and promotes the recruitment of ATRIP–ATR. Ku binds the dsDNA end, may translocate up to dsDNA to ssDNA junction, and recruits DNA-PKcs. When amounts of RPA-covered ssDNA are limited, the concerted phosphorylation of RPA32 and TopBP1 by DNA-PKcs and ATR promotes signal amplification and assembly of a potent ATR signaling complex. IP, immunoprecipitation; P, phosphorylated.