Fig 1: PNUTS associates with the Ku protein complex and regulates DNA-PKcs phosphorylation. (A) MBP-PNUTS pull-down was performed in Xenopus egg extracts. The pull-down product was resolved by SDS-PAGE, and analyzed by mass spectrometry. A number of DNA repair proteins were identified as binding patterns of PNUTS, as shown here with the numbers of peptides. A control pull-down was performed in Xenopus egg extracts using beads without MBP-PNUTS. (B) PNUTS pull-down was performed as in panel A. The extract input, control pull-down, and PNUTS pull-down samples were analyzed by immunoblotting for Ku70, Rad 52 and PNUTS. The ‘Ctr’ sample was a mock pull-down using empty amylose beads. (C) Ku70 IP was performed in the lysates of HeLa cell with or without IR-treatment. The lysate input, control IP, and Ku70 IP products were analyzed by immunoblotting for PNUTS, Ku80 and Ku70. (D) PNUTS IP was performed in HeLa cell lysates. The lysate input, control IP and PNUTS IP products were analyzed by immunoblotting for PNUTS, Ku70 and Ku80. (E) Three segments of PNUTS (N, M and C) were expressed with an N-terminal GST tag, and purified on glutathione beads, as described in Materials and Methods. The beads were incubated in Xenopus egg extracts, re-isolated, and analyzed by immunoblotting for Ku70 and GST. A mock pull-down using empty glutathione beads was performed as control (Ctr). (F) Xenopus egg extracts with or without PNUTS depletion were treated with (dA-dT)70, as indicated. The extract samples were analyzed by immunoblotting for DNA-PKcs phospho-S2056, DNA-PKcs phospho-T2609, DNA-PKcs, PNUTS and H2B. (G) HeLa cells were treated with PNUTS siRNA and 10 Gy IR, as indicated. The cell lysates were analyzed by immunoblotting for DNA-PKcs phospho-S2056, DNA-PKcs phospho-T2609, DNA-PKcs, PNUTS and H2B. (H) PP1 and PNUTS fine-tune the dynamic, and complex pattern of DNA-PKcs phosphorylation. PP1 is required for DNA-PKcs activation after DNA damage, presumably via dephosphorylation of multiple sites within the N, PQR and ABCDE regions. PNUTS modulates the action of PP1 toward S2056 and T2609, as the proper phosphorylation of these sites is required for end processing and ligation.