Chk2 Activation Dependence on Nbs1 after DNA Damage

Abstract

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G 1 arrest. Here we show that the ATMdependent activation of Chk2 by ␥-radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxyterminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.The integrity of genetic information is essential for the life and survival of cells. Genomic lesions arising spontaneously during DNA replication or in response to oxidative metabolism or exposure to radiation or chemical mutagens need to be recognized and repaired. Delay of cell cycle progression at specific checkpoints provides the time necessary to prevent replication and segregation of damaged DNA and to process lesions (reviewed in references 52 and 57). A defective or incorrect activation of the surveillance and repair systems can lead to increased mutagenesis, genomic instability, and ultimately cancer (for a review, see reference 13).The Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare human autosomal recessive diseases (22, 51) exhibiting hypersensitivity to ionizing radiation (IR), immunodeficiency, and increased predisposition to develop cancer. NBS patients, however, do not manifest the hallmarks of AT, i.e., cerebellar ataxia and oculocutaneous telangiectasia. At the cellular level, NBS and AT patients show chromosome instability, hypersensitivity to genotoxic agents, and cell cycle checkpoints defects (1,29,30). These similarities su...

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

“…Several studies showed that the expression of S343A mutant Nbs1 in NBS cells resulted in partial defects in IR-induced S-phase checkpoint activation, radiosensitivity and partial defects in ATM-dependent phosphorylation events including Chk2, SMC1 and FANCD2 (Lim et al, 2000;Buscemi et al, 2001;Nakanishi et al, 2002;Yazdi et al, 2002). Our studies also showed that the MRN complex containing mutant Nbs1 (S343A) failed to stimulate the phosphorylation of Chk2 by ATM although p53 phosphorylation was normal in vitro.…”

“…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).…”

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).…”

The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G2 arrest and cytotoxicity

“…In the present study, suppression of Mre11 had direct effects on temozolomide-induced G 2 arrest. The role of the MRN complex in the G 2 -M checkpoint has been controversial, and whereas it has been shown that NBS cells have a defective G 2 -M transition immediately after a low dose of ionizing radiation (47,48), other reports have suggested that the early G 2 -M checkpoint is independent of NBS1 (49, 50). Whereas Mre11 suppression did not seem to alter the ability of cells to undergo G 2 arrest in the present study, it significantly reduced the ability of the cells to maintain this arrest.…”