Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis

Abstract

Deficiency in Artemis is associated with lack of V(D)J recombination, sensitivity to radiation and radiomimetic drugs, and failure to repair a subset of DNA double-strand breaks (DSBs). Artemis harbors an endonuclease activity that trims both 5'- and 3'-ends of DSBs. To examine whether endonucleolytic trimming of terminally blocked DSBs by Artemis is a biologically relevant function, Artemis-deficient fibroblasts were stably complemented with either wild-type Artemis or an endonuclease-deficient D165N mutant. Wild-type Artemis completely restored resistance to γ-rays, bleomycin and neocarzinostatin, and also restored DSB-repair proficiency in G0/G1 phase as measured by pulsed-field gel electrophoresis and repair focus resolution. In contrast, cells expressing the D165N mutant, even at very high levels, remained as chemo/radiosensitive and repair deficient as the parental cells, as evidenced by persistent γ-H2AX, 53BP1 and Mre11 foci that slowly increased in size and ultimately became juxtaposed with promyelocytic leukemia protein nuclear bodies. In normal fibroblasts, overexpression of wild-type Artemis increased radioresistance, while D165N overexpression conferred partial repair deficiency following high-dose radiation. Restoration of chemo/radioresistance by wild-type, but not D165N Artemis suggests that the lack of endonucleolytic trimming of DNA ends is the principal cause of sensitivity to double-strand cleaving agents in Artemis-deficient cells.

Figure 1.

Chemo/radiosensitivity of Artemis deficient and D165N Artemis mutant fibroblasts. (A) Expression levels of wild-type and D165N mutant Artemis in complemented Artemis-deficient CJ179 cells (CJ Arte⁺, CJ Endo⁻), and in 48BR normal fibroblasts (48BR Arte⁺, 48BR Endo⁻). CJ Vect and 48BR Vect are the respective empty-vector-infected cell lines. Artemis was immunoprecipitated using rabbit polyclonal anti-hArtemis antibody and immunoblotted using chicken polyclonal IgY anti-hArtemis (Abcam). Endogenous Artemis in 48BR cells is below the level of detection. (B–D) Clonogenic survival assays were performed on confluence-arrested serum-starved cells treated with radiation (B), bleomycin (C) or NCS (D). Error bars show SEM from at least three independent experiments.

Figure 2.

Dependence of cellular DSB rejoining on Artemis endonucleolytic activity. (A) γ-H2AX foci were scored in confluence-arrested serum-starved cells following 2 Gy γ irradiation, and the results plotted as the number of foci/cell. (B) 53BP1 foci were scored following 4 Gy γ irradiation. (C) γ-H2AX foci were scored as in (A) following treatment with 6 nM NCS for 1 h. Error bars represent the SEM from three independent experiments except for (B), which shows data from two experiments. (D) PFGE was performed on DNA from confluence-arrested serum-starved normal (48BR) or Artemis-deficient (CJ179) cells that were complemented with wild-type or D165N mutant Artemis. Cells were irradiated and then incubated for the indicated times (in hour) to allow repair before analysis by PFGE. (E) FAR values were plotted after subtraction of the FAR value for unirradiated cells. Fluoromicrographs of a typical repair focus experiment are shown in Supplementary Figure S1. PFGE experiments with NCS were precluded by the large amount of drug required.

Figure 3.

Effect of wild-type/D165N Artemis expression levels on radiosurvival and repair. (A) Artemis cDNA was amplified using real time q-PCR and the relative quantities of Artemis expression for indicated clones (cln) using normal lymphoblastoid cells as calibrator sample and β-actin as endogenous control are plotted. Error bars indicate SEM for 3 independent experiments. All 20 isolated CJ Endo⁻ clones had similar levels of expression. (B) Artemis protein level for selected clones was verified by IP/Western blot. JRL2 = normal lymphoblastoid cells. (C and D) Clonogenic survival assays were performed on confluence-arrested serum-starved clones following exposure to radiation. The data points for CJ Vect and 48BR Vect are same for (C) and (D). Error bars represent SEM for three independent experiments. (E) γ-H2AX foci were scored in confluence-arrested serum-starved high/low wild-type Artemis expressing CJ179 cells. Error bars represent standard deviation for two independent experiments.

Figure 4.

Growth of persistent radiation-induced foci in Artemis-deficient cell lines. (A) Fluorescence microscopy of Artemis-deficient and D165N mutant-complemented CJ179 fibroblasts, showing increase in the size of γ-H2AX foci that persist 12–18 h post-irradiation. (B) Average size of 53BP1 (upper panel, dose of 4 Gy) or γ-H2AX (lower panel, dose of 2 Gy) foci. Focus diameter on fluoromicrographs (A) was measured in one direction parallel to the equatorial plane of the image field. The error bars represent SEM for 35 foci. The arrows are pointing toward large foci.

Figure 5.

PML and γ-H2AX partial co-localization and juxtapositioning after 2 Gy γ-radiation exposure. Double PML/γ-H2AX staining was performed after paraformaldehyde fixation. Confocal analysis of representative cells is shown. Green fluorescence: γ-H2AX (polyclonal anti γ-H2AX, Novus biologicals); red fluorescence: PML (monoclonal anti-PML, Santa Cruz, PGM-3). IR Ctrl = no irradiation.

Figure 6.

Overexpression of D165N mutant Artemis confers high-dose DSB-repair deficiency. (A) γ-H2AX focus assay was performed after 2 Gy γ irradiation. (B) PFGE analysis of normal fibroblasts overexpressing wild-type or D165N mutant Artemis after exposure to 40 Gy γ-rays. The panel displays FAR versus repair time (for 48BR Vect versus 48BR Arte⁺ P > 0.3 at all times; for Vect versus Endo⁻ P = 0.04 at 12 h and P = 0.16 at 18 h, by t-test). (C) Clonogenic survival was determined following treatment with radiation (for Vect versus Arte⁺ P = 0.04 at 4 Gy and P = 0.07 at 6 Gy; for Vect versus Endo⁻ P > 0.3 at all doses). The data for 48BR Vect and 48BR Arte⁺ are same as shown in Figures 1 and 2, and are shown here separately for the sake of clarity and comparison with 48BR Endo⁻. Error bars represent the SEM from three independent experiments except for (A), which shows data from two experiments.