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.

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks

“…Overall, the data suggest that ATM recruitment is not mediated solely by the Nbs1 C-terminus. This is supported by the evidence that ATM makes multiple contacts with the MRN complex (Lee and Paull, 2004;Stracker et al, 2007). What is evident from these studies is the difference in phenotype, which cannot be explained solely by the difference between human and mouse.…”

“…Pellegrini et al (2006) reported a lack of requirement for the C-terminus (22 amino-acid deletion) of Nbs1 for Atm activation and signalling, cell cycle control and radiation survival. In a second mouse model with a closely related deletion (24 amino acids), Nbs1 DC/ DC Atm activation appeared to be normal but downstream signalling through SMC1 was defective (Stracker et al, 2007). In addition, cells from these mice exhibited a defective intra-S-phase checkpoint, but had normal radiation sensitivity and chromosomal stability.…”

“…As support for this they cite data from Lee and Paull (2004) that provide evidence for multiple independent contacts between ATM and the MRN complex. Another mouse model of Nbs1, prepared by the more conventional approach, lacking the C-terminal 24 amino acid (Nbs1 DC/DC ) shared some of the phenotype of NBS cells and that from Nbs1 tr735 (Stracker et al, 2007). Nbs1 DC/DC cells showed normal Atm S1987, p53 and Chk2 phosphorylation, but were defective in structural maintenance of chromosomes (SMC1) and BH3 interacting domain death agonist (BID) phosphorylations.…”

Activation and regulation of ATM kinase activity in response to DNA double-strand breaks

“…Despite the importance of the Nbs1 PIKK interaction motif documented in human cells, Xenopus egg extracts and fission and budding yeast, mice expressing a C-terminal truncated Nbs1 mutant exhibit relatively subtle phenotypes (Difilippantonio et al, 2007;Stracker et al, 2007). In mouse B cells expressing C-terminal truncated Nbs1, ATM interaction with Nbs1 and colocalization of ATM with g-H2AX was similar to normal cells (Difilippantonio et al, 2007).…”

DNA double-strand break repair and development

“…MRN-dependent ATM activation at DNA breaks has been reported to require interaction between ATM HEAT repeats and the C-terminal FXF/Y domain of NBS1 (Falck et al, 2005;You et al, 2005). However, again the in vivo significance of this requires further study as mice engineered to contain a Nbs1 C-terminal truncation were normal for most ATM functions, although some apoptotic defects typical of ATM deficiency were present Stracker et al, 2007).…”