MINIREVIEW Dual role of Nbs1 in the ataxia telangiectasia mutated- dependent DNA damage response Joo-Hyeon Lee and Dae-Sik Lim Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Guseong-D, Yuseong-G, Daejeon, Korea Eukaryotic cells have evolved a signaling pathway that is activated by DNA damage. The primary function of this pathway is to sense DNA strand breaks and then to amplify the initial signal and convey it to down- stream effectors that regulate cell cycle checkpoints and DNA repair [1]. Activation of the DNA damage signaling pathway by DNA double-strand breaks thus leads either to arrest of cell cycle progression and repair of the DNA breaks or, if the damage is too extensive, to death of the cell by apoptosis, thus ensur- ing the maintenance of genomic stability. Dysfunction of this pathway has potentially severe consequences, such as the development of cancer or other conditions related to genomic instability [2]. Among many proteins that participate in the DNA damage signaling pathway, ataxia telangiectasia mutated (ATM) plays a central role. This serine–thre- onine kinase is rapidly activated in response to DNA strand breakage and phosphorylates many targets important in DNA repair or cell cycle checkpoint acti- vation [3,4]. The ATM gene was found to be mutated in individuals with ataxia telangiectasia (AT), a rare autosomal-recessive disorder with pleiotropic clinical phenotypes, including progressive neuronal degener- ation, oculocutaneous telangiectasia, immune dysfunc- tion, cancer predisposition and premature aging. Cells derived from affected individuals show defects in checkpoint control in G1, S and G2 ⁄ M phases of the Keywords ATM; cell cycle; checkpoint control; DNA- damage response; DNA repair; intracellular signaling; Nbs1; nuclear foci; phosphorylation Correspondence D S. Lim, Department of Biological Sciences, Biomedical Research Center, Korea Advanced Institute of Science and Technology, 373–1 Guseoung-D, Yuseong-G, Daejeon 305–701, Korea Fax: +82 42 8692610 Tel: +82 42 8692635 E-mail: daesiklim@kaist.ac.kr (Received 12 December 2005, accepted 8 February 2006) doi:10.1111/j.1742-4658.2006.05191.x The Nbs1 protein associates with Mre11 and Rad50 proteins to form the Mre11–Rad50–Nbs1 complex, which plays an important role in the intracellular signaling pathway activated in response to DNA damage. Mutations in the genes for each of these three components of the Mre11– Rad50–Nbs1 complex result in human diseases characterized by genomic instability. Insight into the functions of Nbs1 in the DNA damage response mediated by the protein kinase, ataxia telangiectasia mutated, has been provided by recent studies. Nbs1 acts both as a downstream target of ataxia telangiectasia mutated in the S-phase checkpoint of the cell cycle as well as an upstream modulator or activator of ataxia telangiectasia mutated in the DNA damage response. Abbreviations AT, ataxia telangiectasia; ATLD, AT-like disorder; ATM, ataxia telangiectasia mutated; BRCT, Brca1 COOH-terminus; Chk2, checkpoint kinase 2; FHA, forkhead associated; IR, ionizing radiation; MRN, Mre11–Rad50–Nbs1; NBS, Nijmegen breakage syndrome; RDS, radioresistant DNA synthesis. 1630 FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS cell cycle, radiation hypersensitivity and an increased frequency of chromosome breakage. A complex of Mre11, Rad50 and Nbs1 proteins (the so-called MRN complex) is another key player in the DNA damage signaling pathway [5]. The MRN com- plex is the primary constituent of nuclear foci that form rapidly after exposure of cells to ionizing radi- ation (IR) and which represent sites of ongoing sensing or repair of DNA double-strand breaks. Hypomorphic mutations of the Nbs1 gene in humans give rise to Nijmegen breakage syndrome (NBS), which is charac- terized by microcephaly, immunodeficiency, chromoso- mal instability, predisposition to cancer and cells that show hypersensitivity to IR and abnormal S-phase checkpoint control [6,7]. Germline hypomorphic muta- tions of the Mre11 gene also result in an AT-like dis- order (ATLD) [8]. The phenotypic similarities among AT, NBS and ATLD indicate that the MRN complex functions in the ATM-dependent signaling pathway activated by DNA damage [9]. In this review, we will discuss recent advances in our understanding of Nbs1 function in the ATM-dependent DNA damage signa- ling pathway. Functional domains of Nbs1 relevant to the DNA damage signaling pathway The human Nbs1 gene was originally cloned by two independent groups with the use of a positional clo- ning approach and direct amino acid sequencing of a 95-kDa protein (p95) that was found to associate with human Mre11 [6,7]. The 754-amino acid protein, p95, encoded by the Nbs1 gene shows a low level of sequence similarity to Saccharomyces cerevisiae Xrs2p. Nbs1 contains a forkhead-associated (FHA) domain and a Brca1 COOH-terminus (BRCT) domain in its NH 2 -terminal region, as well as an Mre11-binding domain and an ATM-binding domain in its COOH- terminal region (Fig. 1). FHA and BRCT domains are often present in eukaryotic nuclear proteins involved in cell cycle checkpoint control or DNA repair. The FHA domain appears to interact with target proteins in a phosphorylation-dependent manner, and the BRCT domain also mediates protein–protein interac- tions. The functional significance of the FHA and BRCT domains in Nbs1 has been indicated by several studies. Cells derived from individuals with NBS that express an Nbs1 protein with a mutation in either of these domains manifest both a defect in IR-induced forma- tion of MRN foci and hypersensitivity to IR [10,11]. Another study found that neither domain contributed to radiation resistance [12], whereas yet another showed that both FHA and BRCT domains were required for S-phase checkpoint control, but that only the BRCT domain was essential for radiation resist- ance [13]. These discrepancies are probably caused by differences in the doses of radiation, in Nbs1 muta- tions, or in Nbs1 expression levels among the studies. The FHA and BRCT domains participate in the interaction of Nbs1 with the phosphorylated histone, c-H2AX, which occurs near sites of DNA strand breakage [14]. Mouse cells that lack c-H2AX do not form Nbs1 foci after exposure to IR, suggesting that the direct interaction of Nbs1 with c-H2AX is required for foci formation by the MRN complex [15]. Cell cycle checkpoint control appears largely intact in the c-H2AX-deficient cells, however, suggesting that foci formation is not directly related to checkpoint func- tion. The MRN-interacting protein, MDC1, was recently shown to contribute to the formation of foci containing Nbs1, 53BP1 and Brca1, on the basis of the observation that down-regulation of MDC1 prevented the formation of such foci in response to IR [15–18]. Whether or not the FHA or BRCT domains of Nbs1 directly interacts with MDC1 remains unclear. Although the functional relevance of the FHA and BRCT domains of Nbs1 appears to differ among stud- ies, we can conclude that both domains are required for recruitment of the MRN complex to DNA lesions (possibly through interaction with c-H2AX or MDC1) and consequent foci formation, as well as for cell sur- vival after exposure to IR. Two serine residues at positions 278 and 343 of human Nbs1 are phosphorylated by ATM on exposure of cells to IR. Cells expressing Nbs1 proteins with mutations at these phosphorylation sites exhibit defect- ive S-phase checkpoint control, suggesting that Nbs1 phosphorylation by ATM is required at least for acti- vation of the S-phase checkpoint in response to IR FHA BRCT Mre11-binding Serine 278 Serine 343 Foci formation Radiation resistance -H2AX binding S phase checkpoint? S phase checkpoint Radiation resistance? MRN complex formation Radiation resistance Cell cycle checkpoint ATM-binding Recruitment of ATM to IR-induced foci 754 0 Fig. 1. Functional domains of Nbs1. The forkhead-associated (FHA) and Brca1 COOH-terminus (BRCT) domains in the NH 2 -terminal region bind to c-H2AX and are required for ionizing radiation (IR)- induced foci formation and radiation resistance. Phosphorylation of Ser278 and Ser343 by ataxia telangiectasia mutated (ATM) is essential for activation of the S-phase checkpoint. The Mre11-bind- ing domain is responsible for binding to Mre11 during formation of the Mre11–Rad50–Nbs1 (MRN) complex. The ATM-binding domain at the COOH-terminus binds to ATM and mediates recruitment of ATM to IR-induced foci. J H. Lee and D S. Lim Role of Nbs1 in ATM-dependent DNA damage signaling FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS 1631 [19–21]. This finding might explain, in part, the failure of cells from patients with AT or NBS to arrest DNA synthesis in response to IR (radioresistant DNA syn- thesis, RDS). However, it remains controversial whether Nbs1 phosphorylation is also required for radiation resistance or IR-induced formation of MRN foci [19–21]. The Mre11-binding domain of human Nbs1 has been localized to amino acids 682–693 in the COOH- terminal region of the protein. Deletion of this region of Nbs1 results in a cellular phenotype virtually identi- cal to that of NBS, including defective formation of MRN foci, radiation hypersensitivity and the impair- ment of checkpoint control [10,22]. These observations suggest that the association of Nbs1 with Mre11– Rad50 is essential for its role in the DNA damage response. In addition, the extreme COOH-terminal region (amino acids 734–754) of Nbs1 mediates the interaction of Nbs1 with ATM and the recruitment of ATM to sites of DNA damage, thereby promoting ATM-dependent signaling [23]. Domains similar to the ATM-binding domain of Nbs1 are also found in ATRIP and Ku80 and are required for the interaction of these proteins with the ATM-related kinase, ATR, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), respectively. This conserved motif found in Nbs1, ATRIP and Ku80 thus appears to be important for DNA damage responses mediated by ATM, ATR and DNA-PKcs. Role of Nbs1 in DNA damage checkpoint control The similar clinical phenotypes of AT, NBS and ATLD suggested that the MRN complex functions in the same DNA damage response pathway as does ATM. Cells from individuals with NBS exhibit partial defects in cell cycle checkpoint control after irradi- ation. Both NBS and ATLD cells fail to transiently inhibit DNA replication in the presence of DNA strand breaks; they thus show the RDS phenotype. This phenotype reflects a failure of intra S-phase checkpoint control and was first characterized in AT cells [24]. This shared RDS phenotype was explained at the molecular level by the observations that ATM phosphorylates Nbs1 on Ser278 and Ser343 and that expression of an Nbs1 protein in which Ser343 is replaced by alanine failed to rescue the S-phase check- point defect in NBS cells. Phosphorylation of the pro- tein SMC1 on Ser957 and Ser966 by ATM, which is necessary for activation of the S-phase checkpoint [25,26], also requires Nbs1 and Brca1. These findings indicate that SMC1 regulation by both ATM and Nbs1 is essential for S-phase checkpoint control. The observation that the RDS phenotype of NBS cells is less pronounced than that of AT cells suggested that the S-phase checkpoint might also be regulated in an Nbs1-independent manner [27]. Indeed, the kinase, checkpoint kinase 2 (Chk2) was shown to be a target of ATM in S-phase checkpoint control, indicating that ATM regulates two parallel pathways to achieve such control. However, phosphorylation of Chk2 by ATM also requires Nbs1 in cells subjected to low-dose irradi- ation (1–2 Gy); it does not require Nbs1 in those exposed to high-dose radiation (> 4 Gy) [10,28]. Together, these various observations suggest that sign- aling by ATM and Nbs1 may differentially influence SMC1 or Chk2 in S-phase checkpoint control, depend- ing on the extent of DNA damage. The contribution of Nbs1 to the G1 and G2 ⁄ M checkpoints remains controversial. NBS cells have been found to be defective in the induction of p53 and in G1 checkpoint control in some studies, but not in others [19,29–32]. A partial defect in G1 checkpoint control, and in the induction of p53 and p21, was apparent in NBS cells exposed to low-dose radiation, but not in those subjected to high-dose irradiation. In addition, the activation of Chk2 in the G2 ⁄ M check- point was found to be impaired in NBS cells after low- dose irradiation [28], but G2 ⁄ M checkpoint control in NBS cells was found to be normal in other studies [10,28,33]. Similar discrepancies have arisen in studies of mice with mutations in the Nbs1 gene [9]. Most human NBS cells express an NH 2 -terminally truncated Nbs1 protein that contains an intact Mre11-binding domain. Differences in the nature of the Nbs1 muta- tion, as well as in the dose of radiation administered to NBS cells, thus probably underlie, at least in part, the discrepancies among studies with regard to the contribution of Nbs1 to the G1 or G2 ⁄ M checkpoints. Dual role of Nbs1 in ATM-dependent DNA damage signaling The rapid localization of the MRN complex to the region of DNA strand breaks, and consequent forma- tion of MRN foci in cells exposed to IR, led to the hypothesis that the MRN complex functions in the sensing of DNA strand breakage and in the activation of the ATM-dependent DNA damage signaling path- way [6,34–36]. However, normal activation of the kin- ase activity of ATM and phosphorylation of the ATM target site (Ser15) in p53 were observed in NBS cells exposed to high-dose radiation [19]. Moreover, the observation that Nbs1 phosphorylation by ATM is required for intra S-phase checkpoint control [19–21] Role of Nbs1 in ATM-dependent DNA damage signaling J H. Lee and D S. Lim 1632 FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS indicated that Nbs1 functions downstream (not up- stream) of ATM in the DNA damage signaling path- way. Nevertheless, several studies showed that ATM activation in response to low-dose irradiation was par- tially defective in NBS and ATLD cells [11,37,38]. Regulation of ATM targets, such as Chk2 and SMC1, has consistently been found to be largely dependent on the MRN complex, even though Nbs1 is not abso- lutely required for Chk2 or SMC1 phosphorylation by ATM in cells exposed to high doses of IR [10,11,25,28,39]. Together, the available data suggest that Nbs1 functions as a downstream target of ATM, as well as a modulator of ATM activity, facilitating ATM activation and ATM-dependent phosphorylation of many downstream substrates in the ATM-depend- ent DNA damage signaling pathway [10,11,39]. Given that ATM is a central player in the cellular response to DNA strand breakage, it is important to understand the mechanisms both by which it is activa- ted and by which it signals to downstream effectors in cells with DNA strand breaks. To date, more studies have focused on the identification of downstream tar- gets of ATM [3] than on the molecular mechanism of ATM activation. Important insight into the mechanism of ATM activation has been provided by a recent study [40] showing that ATM exists as a catalytically inactive dimer or higher-order multimer in the absence of DNA damage. In response to DNA damage, how- ever, ATM undergoes rapid autophosphorylation on Ser1981, resulting in dissociation of the inactive homodimers or multimers to yield active monomers. This autophosphorylation of ATM is triggered in cells within minutes after low-dose irradiation, or even in the presence of two exogenous DNA strand breaks per cell [40]. In addition to DNA damage, chromatin structure-changing molecules are able to induce rapid activation of ATM in the absence of detectable DNA strand breakage [40]. The activation of ATM by DNA strand breakage might thus be mediated, at least in part, by a consequent change in chromatin structure. Further insight into the mechanism of ATM activa- tion has been provided by several recent studies [23,41,42] showing that the MRN complex plays a role both in the recruitment of ATM to the region of DNA strand breakage and in thr activation of ATM in a manner dependent on DNA strand breaks. Whether ATM directly recognizes or senses DNA strand breaks, or whether it is activated directly by such strand breaks, is unclear. Neither ATM immunopre- cipitated from cells, nor purified ATM, was found to be directly activated by DNA stand ends in some stud- ies, whereas purified ATM was shown to bind to DNA ends and its activity to be enhanced by them in others [3]. These contradictory results, with regard to the importance of DNA strand breaks in ATM activation in vitro, suggested the possibility that the status of ATM or cofactors might determine the effect of DNA strand ends on ATM activity. The situation has been clarified by the recent biochemical evidence provided by two studies showing that the MRN complex is important for the activation of ATM [41,42]. The puri- fied recombinant MRN complex was thus found to increase the ability of ATM purified from cells (prob- ably a mixture of monomers and dimers or multimers) to phosphorylate target substrates in the absence of DNA strand breaks [41]. Under these conditions, an MRN complex containing an Nbs1 protein in which Ser343 is replaced with alanine failed to stimulate ATM activity, suggesting that both the presence of Nbs1 and its phosphorylation by ATM are required for stimulation of ATM activity by the MRN complex. In contrast to the lack of a requirement of DNA strand breaks for ATM activation in this latter study, highly purified inactive ATM dimers or multimers were found, in the second study, to be activated by the MRN complex only in the presence of DNA strand ends, resulting in the phosphorylation of downstream targets [42]. Furthermore, both Nbs1 and the unwind- ing of DNA ends by Mre11–Rad50 were found to be sufficient for stimulating ATM activity in vitro. The presence of both the MRN complex and DNA strand breaks thus appeared to result in the efficient convers- ion of inactive ATM dimers or multimers to active monomers. Consistent with this result, the extreme COOH-terminal region of Nbs1 is responsible for association with ATM and the recruitment of ATM to sites of DNA strand breakage [23]. Surprisingly, mutation of the autophosphorylation site of ATM (Ser1981 to alanine) affected neither the dimer-to-monomer transition of ATM nor the stimula- tion of its kinase activity induced by the MRN com- plex in the presence of DNA strand breaks in vitro [42], suggesting that autophosphorylation of ATM on Ser1981 is not required for ATM activation induced by the MRN complex and DNA breaks. This conclu- sion is inconsistent with the previous in vivo finding that autophosphorylation of ATM on Ser1981 is an indicator of ATM activation and monomeric status in cells exposed to radiation [40]. The reason for this con- tradiction remains unknown. It is possible that, in the presence of DNA strand breaks, the MRN complex preferentially binds inactive dimers or multimers of ATM and induces their dissociation to yield partially active monomers that have an increased tendency to undergo autophosphorylation on Ser1981 and thereby generate the fully activated kinase. Autophosphoryla- J H. Lee and D S. Lim Role of Nbs1 in ATM-dependent DNA damage signaling FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS 1633 tion of ATM on Ser1981 may stabilize the active monomer or prevent its oligomerization. Indeed, an ATM protein in which Ser1981 is replaced by aspartic acid, which mimics the autophosphorylated form of the kinase, appears to exist as a monomer [42], although the activity of this mutant was not evaluated. Alternatively, phosphorylation of ATM at other sites, even in the absence of phosphorylation on Ser1981, may be sufficient for the dimer-to-monomer transition triggered by the MRN complex and DNA strand breaks, at least in vitro. Elucidation of the structure of the ATM–MRN complex should provide further insight into the molecular mechanism of ATM activa- tion by MRN and DNA strand breaks. Conclusion Although the role of Nbs1 in the ATM-dependent signaling pathway remains controversial, it is now gen- erally accepted that Nbs1 plays a dual role both as a downstream target and an upstream regulator of ATM (Fig. 2). The role of Nbs1, as an upstream regulator of ATM, appears both to depend on the dose of radi- ation to which cells are exposed as well as to be differ- entially affected by Nbs1 gene mutations. Cells from NBS or ATLD patients, with hypomorphic mutations in the corresponding genes, still manifest partial ATM activity as a result of the expression of truncated Nbs1 or Mre11, respectively; such cells thus exhibit only par- tial checkpoint defects after exposure to low doses of radiation. In cells subjected to low-dose irradiation, Nbs1 is required for both activation of ATM and its recruitment to sites of DNA damage. 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Nature 421, 499–506. 41 Lee JH & Paull TT (2004) Direct activation of the ATM protein kinase by the Mre11 ⁄ Rad50 ⁄ Nbs1 com- plex. Science 304, 93–96. 42 Lee JH & Paull TT (2005) ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308, 551–554. Role of Nbs1 in ATM-dependent DNA damage signaling J H. Lee and D S. Lim 1636 FEBS Journal 273 (2006) 1630–1636 ª 2006 The Authors Journal compilation ª 2006 FEBS . characterized by genomic instability. Insight into the functions of Nbs1 in the DNA damage response mediated by the protein kinase, ataxia telangiectasia mutated,. regard to the contribution of Nbs1 to the G1 or G2 ⁄ M checkpoints. Dual role of Nbs1 in ATM-dependent DNA damage signaling The rapid localization of the MRN