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Figure 1. PP1 and PNUTS are recruited to DNA damage sites in vitro and in vivo. (A) As described in Materials and Methods, beads conjugated with biotin-(dA-dT)70 were incubated in Xenopus egg extracts for 30 min, re-isolated, resolved by SDS-PAGE and analyzed by mass spectrometry. The identified repair proteins and phosphatase subunits are shown, along with the numbers of peptides. A control pull-down was performed in Xenopus egg extracts using beads without oligonucleotides. (B) The biotin-(dA-dT)70 pull-down was performed as in panel A. The extract input, control pull-down and biotin-(dA-dT)70 pull-down were analyzed by immunoblotting for Ku70, PNUTS and PP1γ. (C) HeLa cells transfected with GFP-PNUTS were subjected to laser microirradiation as described in Materials and Methods. The fluorescent signal of GFP and immunofluorescent signal of NBS1 phospho-S343 are shown. (D) HeLa cells were subjected to laser microirradiation. The immunofluorescent signals of PP1β and ATM phospho-S1981 are shown.
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Figure 2. PP1 is required for NHEJ. (A) PP1 depletion was performed in Xenopus egg extracts using beads conjugated with a PP1-binding motif derived from PNUTS, as described in Materials and Methods. Extracts were mock treated with beads alone, depleted of PP1γ (Dep), or PP1γ-depleted and then reconstituted with purified recombinant MBP-PP1γ (Add-back). As in our previous study (35), a non-compatible NHEJ template, linearized with Xho1 and Kpn1, was incubated and the repair efficiency, measured by colony formation, is shown. The level of PP1γ was monitored by immunoblotting, and histone H2B served as a loading control. (B) As in our previous study (35), a compatible NHEJ template linearized with Xho1 was incubated in Xenopus egg extracts with or without PP1 depletion. The repair efficiency, measured by colony formation, is shown. (C) The chromosome-integrated, I-SceI-induced NHEJ reporter cell line was characterized in a previous study (36), and described in Materials and Methods. The cells were transfected with or without PP1γ siRNA as indicated. The repair efficiency, measured by GFP expression, is shown. The cell lysates were analyzed by immunoblotting for PP1γ and H2B. The above experiments were performed at least three times, and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Student's t-test.
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Figure 3. PP1 functions in NHEJ via regulation of DNA-PKcs. (A) The linearized NHEJ template was repaired in Xenopus egg extracts, as in Figure 2B. The extracts were depleted of PP1 (using the PP-binding motif of PNUTS, as in Figure 2A), or treated with a DNA-PKcs inhibitor (NU7441, 20 uM), as indicated. The repair efficiency, measured by colony formation, is shown. Extract samples were analyzed by immunoblotting for DNA-PKcs phospho-S2056, PP1γ and H2B. The experiment was performed at least three times, and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired two-tailed Student's t-test. (B) Xenopus egg extracts with or without PP1 depletion (using the PP-binding motif of PNUTS) were treated with 20 ng/μl of (dA-dT)70 as indicated. Immunoblots of DNA-PKcs phospho-S2056, DNA-PKcs phospho-T2609, DNA-PKcs, PP1β, PP1γ and H2B are shown. (C) In vitro DNA-PKcs kinase assay was performed using XRCC4 as substrate, as described in Materials and Methods. The autoradiography and Coomassie stain of XRCC4 are shown. (D) Xenopus egg extracts were immunodepleted of PP1β, PP1γ or both. The extracts were treated with (dA-dT)70 for 30 min, and analyzed by immunoblotting for DNA-PKcs phospho-S2056, DNA-PKcs, PP1β, PP1γ and H2B. (E) HeLa cells were transfected with control or PP1γ siRNA, treated with 10 Gy IR, and incubated as indicated. Immunoblots of DNA-PKcs phospho-S2056, DNA-PKcs, Smc1 phospho-S957, PP1γ and H2B are shown. (F) Five segments of DNA-PKcs (N, JK, PQR, ABCDE (ABC) and C) were expressed with MBP-tag, and purified on amylose beads, as described in Materials and Methods. The beads were then incubated in HeLa cell lysates, re-isolated, and analyzed by immunoblotting. The ‘Ctr’ sample was a mock pull-down using empty amylose beads. (G–I) Immunoprecipitation of PP1γ (G), PP5 (H) and PP6 (I) was performed in the lysates of HeLa cell with or without doxorubicin treatment. The lysate input, control IP with blank beads, and PP1γ IP products were analyzed by immunoblotting for DNA-PKcs, Ku70 and PP1γ, PP5 and PP6, as indicated.
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Figure 4. PNUTS mediates the DNA damage response and repair. (A) Xenopus egg extracts were supplemented with (dA-dT)70 (DNA damage), WT or W393A PNUTS, as indicated, and incubated for 30 min. The extract samples were analyzed by immunoblotting for Brca1 phospho-S1524, Chk2 phospho-T387, Smc1 phospho-S957, Chk1 phospho-S317 and Chk1. In the lower panel, the relative intensity of Chk1 phospho-S317/Chk1 was measured using NIH ImageJ, and shown by the mean values and standard deviations. (B) HeLa cells were transfected with GFP-PNUTS, and treated with 5 μM doxorubicin for 4 h, as indicated. The cell lysates were analyzed by immunoblotting for ATM phospho-S1981, Chk2 phospho-T68, Chk1 phoshpo-S296, GFP and H2B. (C) HeLa cells with or without GFP-PNUTS expression were treated with doxorubicin for 24 h. Cell death was measured by trypan blue excursion. (D) HeLa cells were treated with PNUTS siRNA and doxorubicin, as indicated. The cell number of each day was normalized to that of the first day for the relative cell viability. (E) HeLa cells with control or PNUTS siRNA were irradiated with 4 Gy IR and incubated as indicated. The cell lysates were analyzed by immunoblotting for γ-H2AX, PNUTS and β-actin. (F, G) HeLa cells treated as in panel E were examined by immunofluorescence for γ-H2AX. The average numbers of γ-H2AX foci were counted (F) and representative images of cells stained for γ-H2AX (in red) and DAPI (in blue) are shown (G). At least 100 cells were analyzed for γ-H2AX foci in panel F.
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Figure 5. PNUTS is required for NHEJ in Xenopus egg extract and human cells. (A) As described in Materials and Methods, HeLa cells were treated with control (Ctr) or PNUTS siRNA, or with PNUTS siRNA and then reconstituted with siRNA resistant GFP-PNUTS (Add-back). NHEJ assay was performed using a GFP-expressing template linearized with Nhe1, as described in Material and Methods. The repair efficiency, measured by GFP expression, is shown. The cell lysates were analyzed by immunoblotting for PNUTS and β-actin. (B) The chromosomal NHEJ reporter cells were transfected with control or PNUTS siRNA. The cell lysates were analyzed by immunoblotting for PNUTS and β-actin. The chromosomal NHEJ assay was performed as in Figure 2C. The repair efficiency, measured by GFP expression, is shown. A minimum of three experiments were carried out in the above panels, and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired two-tailed Student's t-test.
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Figure 6. 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.
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