Signal joint formation in MRN-deficient abl pre–B cells. (a) Schematic of the pMX-DEL^SJ retroviral recombination substrate. All components of the retrovirus are as indicated in the Fig. 1 legend. The signal end (SE) and signal joint (SJ) are indicated. (b) Southern blot analysis of EcoRV-digested genomic DNA from Nbs1^m/m (line 675.3), Mre11^ATLD1/ATLD1 (line 48.1), and Ku70^−/− (line 0.2) abl pre–B cells containing the pMX-DEL^SJ (DEL^SJ) retroviral recombination substrate that had been treated with STI571 for the indicated number of hours). (c) PCR analysis of pMX-DEL^SJ signal joints generated in WT:DEL^SJ, Atm^−/−:DEL^SJ, Mre11^ATLD1/ATLD1:DEL^SJ, and Nbs1^m/m:DEL^SJ abl pre–B cells treated with STI571 for 72 h. PCR was performed using primers pB and pC (a), and PCR products were either not digested (−) or digested with ApaLI (+). Products were visualized by ethidium bromide staining, and undigested (SJ) and digested (CP) signal joint products are indicated. The data presented are representative of at least two experiments.

Rag-DSB-mediated Atm activation in MRN-deficient cells. (a) Western blot analysis for phospho–KAP-1 (α–p–KAP-1) and KAP-1 (α–KAP-1) in Artemis^−/− (line 5.1), Nbs1^m/m:Artemis^−/− (line 37.1), and Mre11^ATLD1/ATLD1:Artemis^−/− (line 64.1) abl pre–B cell lines treated with STI571 for 96 h and either DMSO (−) or the Atm inhibitor (iAtm) KU-55933 (+). (b) Percentage of Artemis^−/− (line 5.3), Nbs1^m/m:Artemis^−/− (line 37.4), and Mre11^ATLD1/ATLD1:Artemis^−/− (line 61.2) abl pre–B cells with one, two, or three to five nuclear γ-H2AX foci after treatment with STI571 for 24 h and either DMSO (−) or the Atm inhibitor (iAtm) KU-55933 (+). The total number of cells analyzed for each genotype was 500 and p-values were calculated using a two-tailed Fisher's Exact test. (c) NF-κB EMSA of nuclear lysates from Artemis^−/−, Atm^−/−: Artemis^−/−, and two independent Nbs1^m/m:Artemis^−/− and Mre11^ATLD1/ATLD1:Artemis^−/− abl pre–B cell lines treated with STI571 for 36 h. NFY EMSA is shown as a control. The data presented are representative of at least two experiments.

Thymocyte development in Nbs1^m/m mice. (a) Number of thymocytes in Atm^−/− (n = 16) and Nbs1^m/m (n = 9) mice at the indicated stages of development, expressed as a percentage of the number of thymocytes found at each stage in WT Atm^+/+ (n = 12) and Nbs1^+/+ (n = 3) littermate controls. Calculations were done using mean numbers of thymocytes from mice of each genotype and propagated error. Error bars represent SEM. DN (double negative), CD4⁻CD8⁻; DP, CD4⁺CD8⁺; SP, single positive. (b) Representative flow cytometric analysis of TCR-β expression on CD4⁺CD8⁺ DP thymocytes from WT, Nbs1^m/m, and Atm^−/− mice. Numbers indicate percentage of DP cells that are TCR-β^int. Results are representative of the analyses of at least three mice.

Rearrangement of pMX-INV in Nbs1^m/m and Mre11^ATLD1/ATLD1 abl pre–B cells. (a) Schematic of the pMX-INV retroviral recombination substrate, rearrangement intermediates, and products. The retroviral packaging signal (ψ), GFP complementary DNA, and IRES-human CD4 (I-hCD4) cassette are labeled. The viral LTRs (open arrows) and the RSs (open triangles) are shown. The nonrearranged (NR) pMX-INV and pMX-INV coding ends (CE), coding joint (CJ), and hybrid joint (HJ) are indicated. The relative position of the pA, pB, and pC oligonucleotides, the C4 probe (bar), and EcoRV (E) and NcoI (N) endonuclease restriction sites are shown, as are the expected sizes of hybridizing fragments. (b and c) Southern blot analysis of EcoRV–NcoI-digested (b) or EcoRV-digested (c) genomic DNA from WT, Atm^−/−, Mre11^ATLD1/ATLD1 (Mre11^A/A), and Nbs1^m/m abl pre–B cell clones containing the pMX-INV (INV) retroviral recombination substrate that had been treated with STI571 for the indicated time (hours). The abl pre–B cell clones analyzed each have single pMX-INV integrants and were derived from parental lines as indicated (parental line, clone number). Expected sizes for bands generated by nonrearranged pMX-INV (NR), coding joints (CJ), hybrid joints (HJ), and coding ends (CE) are indicated. (d and e) Southern blot analysis of EcoRV–NcoI-digested (d) or EcoRV-digested (e) genomic DNA from WT (line A70.2), Atm^−/− (line 2F), Mre11^ATLD1/ATLD1 (Mre11^A/A; line 48.1), and Nbs1^m/m (line 737.3) abl pre–B cells containing the pMX-INV (INV) retroviral recombination substrate that had been treated with STI571 for the indicated time (hours). (f) Quantification of rearrangement products from blots in d and e. Products are expressed as a percentage of the total fraction of pMX-INV substrates that had initiated V(D)J recombination (CJ + HJ + CE) to normalize for differences in cleavage efficiency between the cell lines. The data presented are representative of at least two experiments.

Atm activation by Rag DSBs. (a) Southern blot analysis showing Jκ1 and Jκ2 coding ends (CE) generated in Rag2^−/−, Artemis^−/−, and Artemis^−/−:Atm^−/− abl pre–B cells treated with STI571 for the indicated times (hours). SacI- and EcoRI-digested genomic DNA was hybridized to the JκIII probe. The bands corresponding to the IgL-κ locus in the germline configuration (GL) and Jκ1 and Jκ2 CEs are indicated. (b) Western blot analysis of phospho-KAP-1 (α-p-KAP-1) and KAP-1 (α-KAP-1) from STI571-treated cells shown in a. (c) Quantification of γ-H2AX nuclear foci in the cells from a after 24 h of STI571 treatment. Shown is the percentage of cells containing one, two, or three to five γ-H2AX foci. The total number of cells analyzed for each genotype was 500 and p-values were calculated using a two-tailed Fisher's Exact test. Note that γ-H2AX foci were not detected in any of the STI571-treated Rag^−/− abl pre–B cells. The data presented are representative of at least two experiments.

Atm inhibition in MRN-deficient cells leads to increased coding end accumulation. Southern blot analysis of EcoRV-digested genomic DNA from WT, Atm^−/−, and two Mre11^ATLD1/ATLD1 (labeled as Mre11^A/A) and Nbs1^m/m abl pre–B clones containing pMX-INV. Cells were treated with STI571 for the indicated time (hours) and either DMSO (−) or the Atm inhibitor (iAtm) KU-55933 (+). Expected sizes for bands generated by nonrearranged substrate (NR), coding joints (CJ), hybrid joints (HJ), and coding ends (CE) are indicated. The data presented are representative of at least two experiments.

V(D)J recombination defects in MRN-deficient lymphocytes in vivo. (a) Schematic of Rag cleavage at the Jα56 gene segment on the TCR-α^sJ allele. The gene segments are shown as black rectangles, RSs are open triangles, and the Cα constant region is the gray rectangle. The StuI restriction sites (S) and the Cα1 probe (black bar) used for analysis are also shown. (b) Southern blot analysis of TCR-α rearrangement in WT, Atm^−/−, Nbs1^m/m, and Mre11^ATLD1/ATLD1 thymocytes, all on the TCR-α^sJ/sJ background. The expected sizes for germline TCR-α^sJ allele (*) and Jα56 coding ends (Jα56 CE) are indicated. Genomic DNA was digested with StuI and probed with the Cα1 probe. Hybridization to a Rag-2 probe (PR2) is shown as a DNA loading control. Results are representative of three experiments. (c) PCR analysis of Vδ5DδJδ1 coding joints (Vδ5 CJ), Vδ5Dδ1 hybrid joints (Vδ5 HJ), Vβ14DβJβ2.7 coding joints (Vβ14 CJ), and Vβ14Dβ2 hybrid joints (Vβ14 HJ) in WT, Atm^−/−, Nbs1^m/m, and Mre11^ATLD1/ATLD1 thymocytes. (d) PCR analysis of Vκ6-23 to Jκ1 coding joints (Vκ6-23 CJ) and hybrids joints (Vκ6-23 HJ) in WT, Atm^−/−, Nbs1^m/m, and Mre11^ATLD1/ATLD1 splenocytes. The IL-2 gene PCR is shown as a DNA quantity control. The data presented are representative of analyses of at least two mice of each genotype.