DNA structure-specific priming of ATR activation by DNA-PKcs

Abstract

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

Figure 1.

DNA damage signal activation in human cell-free extracts. (A) The duplex DNA substrates are blunt ended and 573 bp long. The gDNA contains a 68-nt single-stranded gap. (B) The gDNA is refractory to digestion with SpeI. (C) gDNA-specific phosphorylation of RPA32 and Chk1. Nuclear extracts were incubated without DNA (lane 1), with duplex DNA (lane 2), or with gDNA (lane 3). The indicated proteins were analyzed by Western blotting. (D) Proteins bound to biotinylated DNA were pulled down with streptavidin-coated beads and detected by Western blotting. The dsDNA and gDNA substrates are represented schematically. Biotin (black circles) and streptavidin-coated beads (dented gray circles) are shown.

Figure 2.

The juxtaposition of DNA ends and an ssDNA gap triggers activation of the DNA damage signal. (A) Nuclear extracts were incubated for the indicated time periods with open circular plasmid DNA (lanes 1–5), linear duplex DNA (lanes 6–10), gap circular DNA (lanes 11–15), or gapped linear duplex DNA (lanes 16–20). The indicated proteins were analyzed by Western blotting. (B) ATR activation depends on accessible DNA ends. (lanes 1–3) dsDNA and gDNA were biotinylated at both ends and incubated with nuclear extracts. (lanes 4–6) Biotinylated DNA substrates (5 nM) were preincubated with 30 nM streptavidin for 15 min at 37°C before addition of nuclear extracts. (lanes 7–9) Unmodified DNA substrates were preincubated with streptavidin before addition of nuclear extracts. The DNA substrates are represented schematically. Biotin (black circles) and streptavidin (gray circles) are shown.

Figure 3.

Concerted activation of DNA damage signaling by DNA-PKcs, ATM, and ATR. (A) DNA-PKcs, ATM, and ATR bind to the biotinylated DNA substrates. DNA structures biotinylated at one DNA end were coupled to streptavidin-coated beads and then incubated with nuclear extracts for 10 min at 20°C, in the absence or presence of the DNA-PKcs inhibitor IC86621 (100 µM), the ATM inhibitor KU-55933 (10 µM), or the ATR inhibitor ETP-46464 (1 µM), as indicated. DNA-bound DNA-PKcs, ATM, and ATR were pulled down with streptavidin-coated beads, resolved by SDS-PAGE, and detected by Western blotting. (B) DNA-bound TopBP1 and Chk1 were isolated and detected as described in A. (C) Nuclear extracts prepared from cells treated with control shRNA or ATR shRNA were incubated with the indicated biotinylated DNA substrates, as described in A. ATR, DNA-PKcs, and Chk1 proteins were pulled down with streptavidin-coated beads, resolved by SDS-PAGE, and detected by Western blotting. (D) Nuclear extracts from cells treated with control shRNA or RPA shRNA were incubated for the indicated time periods with gDNA and probed for the indicated proteins by Western blotting. iDNA-PKcs, inhibitor of DNA-PKcs; iATM, inhibitor of ATM; iATR, inhibitor of ATR.

Figure 4.

DNA-PKcs is essential for DNA damage signal activation. (A) Nuclear extracts prepared from cells treated with control shRNA (shControl) or DNA-PKcs shRNA were incubated without DNA, with dsDNA, or with gDNA, and the indicated proteins were detected by Western blotting. (B) Nuclear extracts from HCT116 and HCT116 DNA-PKcs^−/− cells were incubated with gDNA, and HCT116 DNA-PKcs^−/− nuclear extracts were complemented with increasing amounts of DNA-PK purified from HeLa cells (0.025, 0.05, 0.1, 0.2, and 1 U/µl). (C) Nuclear extracts from cells treated with control shRNA or Ku70 shRNA were incubated for the indicated time periods with gDNA and probed for the indicated proteins by Western blotting. (D) Biotinylated DNA substrates were incubated with nuclear extracts in the absence or presence of IC86621 and pulled down, and the indicated DNA-bound proteins were analyzed by Western blotting. (E) Biotinylated DNA substrates were incubated with nuclear extracts from cells treated with control shRNA or ATR shRNA, isolated, and probed for the indicated proteins.

Figure 5.

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.