The carboxy terminus of NBS1 is required for induction of apoptosis by the MRE11 complex

Abstract

The MRE11 complex (MRE11, RAD50 and NBS1) and the ataxia-telangiectasia mutated (ATM) kinase function in the same DNA damage response pathway to effect cell cycle checkpoint activation and apoptosis. The functional interaction between the MRE11 complex and ATM has been proposed to require a conserved C-terminal domain of NBS1 for recruitment of ATM to sites of DNA damage. Human Nijmegen breakage syndrome (NBS) cells and those derived from multiple mouse models of NBS express a hypomorphic NBS1 allele that exhibits impaired ATM activity despite having an intact C-terminal domain. This indicates that the NBS1 C terminus is not sufficient for ATM function. We derived Nbs1(DeltaC/DeltaC) mice in which the C-terminal ATM interaction domain is deleted. Nbs1(DeltaC/DeltaC) cells exhibit intra-S-phase checkpoint defects, but are otherwise indistinguishable from wild-type cells with respect to other checkpoint functions, ionizing radiation sensitivity and chromosome stability. However, multiple tissues of Nbs1(DeltaC/DeltaC) mice showed a severe apoptotic defect, comparable to that of ATM- or CHK2-deficient animals. Analysis of p53 transcriptional targets and ATM substrates showed that, in contrast to the phenotype of Chk2(-/-) mice, NBS1(DeltaC) does not impair the induction of proapoptotic genes. Instead, the defects observed in Nbs1(DeltaC/DeltaC) result from impaired phosphorylation of ATM targets including SMC1 and the proapoptotic factor, BID.

Figure 1. Generation of Nbs1^ΔC/ΔC mice

a, The C-terminal sequence of NBS1^ΔC is shown compared with wild-type NBS1 (conserved residues in bold),. b, PCR analysis and sequencing of complementary DNA confirmed splicing from exon 14 to 16 and a nonsense mutation that results in the truncation of the 24 C-terminal amino acids. c, Immunoblotting (IB) showed increased mobility of NBS1^ΔC in Nbs1^ΔC/ΔC MEFs. d, Immunoprecipitation (IP) of NBS1 and IB for NBS1 (top) and MRE11 (bottom) from MEFs of the indicated genotype. IR, ionizing radiation.

Figure 2. Cellular phenotypes of Nbs1^ΔC/ΔC

a, G1/S checkpoint analysis in MEFs of the indicated genotype. Cells were mock (white), 5 Gy IR (grey) or 10 Gy IR (black) -treated (n = 3; error bars, s.d.). b, G2/M checkpoint analysis in MEFs of the indicated genotype. Cells were mock (white), 2 Gy IR (grey) or 10 Gy IR (black) -treated (n = 3; error bars, s.d.). c, Intra-S-phase checkpoint in wild-type (diamond), Nbs1^+/ΔC (square), Nbs1^ΔC/ΔC (triangle), Nbs1^ΔB/ΔB (circle) or Atm^−/−(cross) MEFs. d, IB of SMC1-S957-p and SMC1, in MEFs of the indicated genotype. e, IB of ATM-S1981-p and ATM in MEFs after IR treatment.

Figure 3. Apoptotic phenotypes of Nbs1^ΔC/ΔC

a, Representative TUNEL stained sections of small intestines from the indicated genotype (top). Haematoxylin and eosin (H&E)-stained sections of testes (bottom). b, Cleaved caspase-3 staining of thymi post IR treatment. c, Dose response of thymocytes post IR treatment. Triplicate results from 2 Nbs1^ΔC/ΔC (open and closed squares) and 2 wild type (open and closed diamonds) animals are shown (n = 3, error bar = s.d.). d, Thymocyte apoptosis in the indicated genotypes (n, number of animals; error bar, s.d. of triplicate results). P-values (Wilcoxon rank sum test) are: P(Nbs1^ΔC/ΔC versus wild type) = 2.96 × 10⁻⁷; P(Nbs1^ΔC/ΔC versus Atm^−/−) = 0.35; and P(Nbs1^ΔC/ΔC vs. Nbs1^ΔC/ΔC Atm^−/−) = 2.311 × 10⁻⁵.

Figure 4. Apoptotic signalling in Nbs1^ΔC/ΔC

a, Quantitative PCR analysis of p53-dependent proapoptotic genes. Induction of Bax and Puma from a representative experiment performed in triplicate is shown (error bars, s.d.). Mock-treated (white) or IR-treated (black) thymocytes, 8 h post 5 Gy IR. b, Western blot analysis of p53-S18, p53, phosphorylated BID (S61, S78), BID, and actin in thymocytes after 5 Gy of IR at the indicated times post treatment. c, Western blot analysis of CHK2 hyperphosphorylation in thymocytes at the indicated times post 5 Gy IR.