Artemis is phosphorylated in vivo after exposure of MCF-7 cells to IR or UV irradiation. (A) Schematic of Artemis indicating locations of S/TQ motifs. Asterisks above or below the schematic indicate motifs conserved or not conserved in humans and mice, respectively. aa, amino acids. (B) Immunoblotting shows the absence of Artemis in Artemis-deficient P11 cells. The asterisk indicates a nonspecific loading control. (C) Treatment with either IR or UV results in phosphorylation of Artemis (Artemis-P). Artemis was immunoprecipitated from extracts of cells exposed to IR (10 Gy) or UV (20 J/m²) and subsequently incubated for 2 h (lanes 1 and 4). The same immunoprecipitate was incubated with alkaline phosphatase (lanes 2 and 5) or treated with alkaline phosphatase in the presence of the phosphatase inhibitor Na₃VO₄ (lanes 3 and 6). The immunoprecipitate from untreated cells (UT) is shown in lane 7. (D) Phosphorylation of Artemis as a function of IR dose examined 2 h after treatment. (E) Kinetic analysis of the phosphorylation of Artemis after the cells were exposed to IR (10 Gy) for times ranging from 5 min (5m) to 17 h. (F) Phosphorylation of Artemis as a function of UV dose examined 2 h after treatment. (G) Kinetic analysis of the phosphorylation of Artemis after exposure of cells to UV (20 J/m²).

Artemis interacts with checkpoint proteins and is phosphorylated by ATM and ATR. (A, top blot) Artemis antibodies coimmunoprecipitate ATM, BRCA1, Rad50, Nbs1, and Mre11 from HeLa extracts. Protein A-Sepharose beads were used as a control. IP, immunoprecipitation; IgG, nonspecific immune serum. Immunoblotting (IB) was performed using antibodies to the indicated proteins. (A, lower blot) Reciprocal coimmunoprecipitation of Artemis by ATM antibodies. ATM+I, incubation of the extracts with DNase I prior to coimmunoprecipitation. (B) Depletion of Artemis in HeLa cells by transfection of siRNA eliminates the coimmunoprecipitation of Rad50 by Artemis antibodies. α-Artemis IP, immunoprecipitation with anti-Artemis antibodies; Pre., preimmune serum; Imm., Artemis antiserum. (C) Immunoprecipitation (IP) kinase assay shows that Artemis was phosphorylated by both ATM and ATR but by kinase-dead (kd) variants of the enzymes. The top shows an autoradiogram of Artemis phosphorylation by ATM or ATR. The bottom shows immunoblots of immunoprecipitated kinases used in the assays.

Artemis is required for a normal DNA damage-induced G₂/M cell cycle checkpoint. (A) Analysis of the G₂/M checkpoint after IR. Fluorescence-activated cell sorting profile of Artemis-depleted and control HEK293 cells stained with propidium iodide and anti-phospho-histone H3 (P-H3) at the indicated times after IR irradiation (6 Gy). The percentages of cells in the G₁/S and G₂/M phases are shown. (B) Graphical analysis of P-H3 staining indicates a defect in the G₂/M checkpoint in Artemis-depleted cells. The fraction of P-H3-positive cells is expressed as a percentage of that measured at the 0-h time point. (C) P-H3 accumulation is not due to differential exit from mitosis. Values of P-H3 levels for the untreated sample (UT) were set at 1.0, and results for the other samples are shown as a percentage of this value. Nocodazole was added 30 min after IR treatment, and data were collected after 16 h. (D) Comparison of the cell cycle fractions of Artemis- and DNA-PKcs-depleted cells 16 h after IR (graphs at the top). The levels of cyclin B in the same cells are shown (middle blot). Immunoblotting showing depletion of DNA-PKcs by siRNA in HEK293 cells is shown (bottom blot). LC, loading control.

Artemis is not essential for NHEJ. (A) Immunodepletion of Artemis from HeLa whole-cell extracts as determined by immunoblotting. HeLa whole-cell extract (lane 1); HeLa whole-cell extract precipitated with protein A-Sepharose beads only (lane 2); whole-cell extract precipitated with preimmune serum (Pre.) (lane 3); and Artemis-depleted whole-cell extract (depleted with Artemis antiserum) (lane 4). (B) Immunodepletion of Artemis from HeLa cell extracts does not affect end joining of linearized plasmid DNA. Ethidium bromide-stained agarose gels show the results of end-joining assays. Plasmid DNA was linearized by digestion with BamHI. The positions of monomer (M) and dimer (D) plasmids are indicated to the left of the gels. DNA only was used as a control (C) in lane 1. Assays shown in lanes 2 to 4 were performed with whole-cell extracts treated as described above for panel A. DNA size markers (S) are shown in lane 5. IgG, nonspecific immune serum. Controls (lanes 6 to 9) show that wortmannin (Wort.) and antibodies to DNA-PKcs inhibit the rejoining reaction. (C) DSB rejoining is normal in Artemis-deficient cells. P11 cells expressing Artemis, the simian virus 40-transformed human cell line XP2OS (considered the wild type [wt] in these experiments), and MO59J (DNA-PKcs-deficient) cells were compared as a function of IR dose for DNA rejoining by pulsed-field gel electrophoresis. (D) DSB rejoining is normal in HEK293 cells depleted of Artemis by siRNA. Depletion of Artemis by siRNA is shown by immunoblotting. Con., control; Art., Artemis. (E) DSB rejoining examined as a function of time of incubation. The + indicates the value at the 4-h time point for the MO59J cell line.

Artemis is phosphorylated in vivo by DNA-PKcs after IR. (A) Phosphorylation of Artemis in vivo after exposure of cells to IR is deficient in MO59J cells. MO59J and MO59K cells were treated with IR (10 Gy), incubated for 1 h prior to extract preparation, and compared with untreated cells (UT). Caff., caffeine. (B) Depletion of DNA-PKcs by siRNA shows that DNA-PKcs and a caffeine-sensitive kinase(s) participate in the phosphorylation of Artemis after IR. LC, loading control. The presence (+) or absence (−) of control and DNA-PKcs siRNA, caffeine, and IR irradiation are indicated above the blots. (C) Phosphorylation of Artemis after IR treatment is inhibited by wortmannin (Wor.) in MCF-7 cells, but not by the Chk1 inhibitor UCN-01 (UCN.). (D) Artemis antibodies coimmunoprecipitate DNA-PKcs with and without exposure to IR in HCT116 cells. (E) DNA-PKcs interacts with phosphorylated and unphosphorylated forms of Artemis as determined by coimmunoprecipitation (IP) with DNA-PKcs antibodies. HEK293 cells stably transfected with pDEST27-Artemis (expressing GST-Artemis) were exposed to IR and incubated for 1 h. Protein A-Sepharose beads were used as a control.

Phosphorylation of Artemis is partially dependent upon ATM after IR irradiation and upon ATR after UV irradiation. (A) Artemis is phosphorylated in ATM-deficient cells (GM9607) after IR irradiation. UT, untreated cells. (B, top blot) Depletion of ATM expression in MO59J cells by siRNA. LC, loading control. (B, bottom blot) Phosphorylation of Artemis in MO59J cells depleted of ATM is affected after IR irradiation, but not after UV irradiation. (C, top blot) Elimination of ATR expression by infection of ATR^flox/− cells with adenovirus expressing Cre recombinase. (Bottom blot) Phosphorylation of Artemis in cells depleted of ATR is affected after UV irradiation, but not after IR irradiation.

Artemis is not involved in established G₂/M checkpoint pathways. Where not indicated, the IR dose was 20 Gy and the UV dose was 50 J/m². All experiments were performed 2 h after irradiation. (A) Phosphorylation of Chk1 (phospho-Ser345) and Chk2 (phospho-Thr68) are unaffected in Artemis-depleted cells after IR or UV irradiation. UT, untreated cells; LC, loading control. (B) Phosphorylation of H2AX is unaffected in Artemis-depleted cells. Numbers shown after the indicated treatment specify the dosage in grays (IR) or joules per square meter (UV). (C) Phosphorylation of Artemis is unaffected in Rad17-depleted cells. Depletion of Rad17 in RAD17^flox/− cells is shown after infection with adenovirus expressing Cre recombinase. (D) Phosphorylation of Artemis is unaffected in 53BP1-depleted cells.