Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest

Abstract

Cell lines from Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) patients show defective S phase checkpoint arrest. In contrast, only A-T but not NBS cells are significantly defective in radiation-induced G1/S arrest. Phosphorylation of some ATM substrates has been shown to occur in NBS cells. It has, therefore, been concluded that Nbs1 checkpoint function is S phase specific. Here, we have compared NBS with A-T cell lines (AT-5762ins137) that express a low level of normal ATM protein to evaluate the impact of residual Nbs1 function in NBS cells. The radiation-induced cell cycle response of these NBS and 'leaky' A-T cells is almost identical; normal G2/M arrest after 2 Gy, intermediate G1/S arrest depending on the dose and an A-T-like S phase checkpoint defect. Thus, the checkpoint assays differ in their sensitivity to low ATM activity. Radiationinduced phosphorylation of the ATM-dependent substrates Chk2, RPAp34 and p53-Ser15 are similarly impaired in AT-5762ins137 and NBS cells in a dose dependent manner. In contrast, NBS cells show normal ability to activate ATM kinase following irradiation in vitro and in vivo. We propose that Nbs1 facilitates ATM-dependent phosphorylation of multiple downstream substrates, including those required for G1/S arrest.

Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability

“…This inadequacy is also observed in AT hybrid cells (AT cells6normal cells) (Komatsu et al, 1989). In addition to the defect in intra-S phase checkpoint control, disruption to G2 and G1 checkpoints after irradiation is also observed in NBS cells (Shiloh, 1997;Ito et al, 1999;Buscemi et al, 2001;Girard et al, 2002).…”

“…This is exhibited by the evidence that impairment of the G2 checkpoint is due to the delay in CHK2 phosphorylation and the introduction of full length NBS1 into NBS cells restores this delay to the level of wild type cells (Buscemi et al, 2001). On the other hand, Girard et al (2002) demonstrated dose dependence for these checkpoint controls. Defects in the G1 checkpoint and also in the induction of p53/p21 are observed in NBS cells exposed to low doses, but these are not apparent in cells exposed to high doses of ionizing radiation.…”

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

“…The Mre11/Rad50/Nbs1 (MRN) complex has been shown by several groups to be required for the ATM signaling pathway (Lim et al, 2000;Buscemi et al, 2001;Girard et al, 2002;Nakanishi et al, 2002;Yazdi et al, 2002;Uziel et al, 2003). Most of these studies were performed using cells derived from patients with Nijmegen Breakage Syndrome (NBS) or Ataxia-TelangiectasiaLike-Disorder (ATLD), which are caused by hypomorphic alleles of the Nbs1 or Mre11 genes, respectively (Carney et al, 1998;Varon et al, 1998;Stewart et al, 1999).…”

“…The MRN complex was first considered to be in the same pathway with ATM because both NBS and ATLD patients exhibit similar clinical and cellular phenotypes compared to A-T patients, including chromosomal instability, radiation sensitivity and defects in cell cycle checkpoints. Girard et al (2002) showed that phosphorylation of p53 on Ser15 was impaired in NBS cells following exposure to low doses of ionizing radiation (IR), suggesting that Nbs1 may be required for ATMdependent phosphorylation of substrates in cells. Uziel et al (2003) also showed that ATM-dependent phosphorylation of p53, Chk2 and Mdm2 was defective in ATLD cells after treatment with neocarzinostatin (NCS), a radiomimetic agent.…”

Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation

“…Whereas the checkpoint functions of the MRN complex lie downstream of ATM, being regulated by the ATM-mediated phosphorylations of Nbs1 (Shiloh, 2003), the involvement of ATM in the accumulation of the MRN proteins at the sites of DNA damage seems less likely (Mirzoeva and Petrini, 2001). In addition, several studies of cellular responses to IR report suboptimal phosphorylation of some ATM targets in cells deficient in Nbs1 (Buscemi et al, 2001;Girard et al, 2002;Yazdi et al, 2002;Gatei et al, 2003;Lee et al, 2003), indirectly suggesting a potential role of the MRN complex in modulating some aspect(s) of ATM function. Thus, despite both ATM and the MRN complexes playing critical early roles in response to DSBs and having been considered as candidate sensors of DNA damage (Petrini, 2000;Durocher and Jackson, 2001;Khanna and Jackson, 2001), the exact temporal order and molecular basis of the interplay between ATM and MRN remain to be clarified.…”