SUMMARY The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a divalent MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.The eukaryotic DNA damage response represents a series of highly integrated and tightly regulated pathways that coordinate DNA repair, cell cycle, and homeostatic responses to abnormal DNA structures arising endogenously or following exposure to extrinsic genotoxic stimuli. Central to the DNA damage response are a pair of structurally and functionally related protein kinases, designated ATM 2 (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) belonging to the phosphoinositide 3-kinase-related kinase (PIKK) gene superfamily. ATM and ATR share a conserved carboxyl-terminal catalytic domain and display highly overlapping substrate specificities in vitro (1-3). Substrates for ATM and ATR include the p53 and BRCA1 tumor suppressors among many other proteins involved in cell cycle checkpoint activation, DNA repair, and transcriptional regulation (1, 3). Mutations in ATM cause the cancer susceptibility-neurodegeneration syndrome, ataxia-telangiectasia (1, 4). ATM-deficient cells are grossly defective in the ionizing radiation (IR)-induced G 1 /S, intra-S phase, and G 2 /M checkpoints and are profoundly sensitive to IR and other agents that induce DNA double-strand breaks (DSBs) (1). Although structurally and functionally related to ATM, the major functions of ATR pertain to its roles in DNA replication (5). ATR prevents premature firing of DNA replicons, couples the completion of S phase to mitosis, and is required for chromosome maintenance and stabilization of stalled DNA replication forks (6 -10). Although null mutations in ATR are lethal, hypomorphic splicing mutations that reduce ATR protein levels are associated with a rare congenital condition known as Seckel's syndrome (11-13).The catalytic activity of ATM is rapidly up-regulated in response to IR and other DSB-inducing agents. Catalytic activation involves the transautophosphorylation of inactive, dimeric ATM on Ser-1981, followed by dissociation into active monomers (14). The trimeric complex of MRE11, RAD50, and NBS1 (MRN) facilitates ATM activation and ATMdependent substrate phosphorylation through recruitment of ATM to DSBs and/or orientation of the ATM catalytic domain (15-18). Recent studies suggest that the recruitment of ATM is mediated by the carboxyl terminus of NBS1 (19,20). Although ATR isolated from DNA-damaged cells does not show enhanced kinase activity, its recruitment to regions of stalled DNA replication is regulated through binding to the ATRinteracting protein (ATRIP) and replication protein A (RPA) (19,21). Among many key substrates for ATR and ATM are the checkpoint effector kinases, CHK1 and CHK2, which are phosphorylated by ATM and ATR in response to DSBs and DNA replication stress, respectively (6,(22)(23)(24). CHK1 and CHK2 promote checkpoint arrest through phosphorylation and inactivation of CDC25 family phosphatases (25-28).Virus infection can also elicit ATM-dependent...

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G 2 /M cell cycle arrest, and apoptosis in HeLa cells. RPAdeficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G 2 /M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea-or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMPresponse element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways. Replication protein A (RPA)1 is a trimeric complex composed of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication in all organisms (1). RPA represents the major cellular single-stranded DNA (ssDNA) binding activity in eukaryotic cells and coats ssDNA filaments stoichiometrically in vitro (1). Through its binding and stabilization of ssDNA, RPA facilitates the unwinding and destabilization of double-stranded DNA, which represents a critical step during DNA replication, recombination, and repair. The major DNA binding activity of RPA resides within the 70-kDa RPA1 subunit, which contains a centrally positioned, high affinity, bipartite DNA-binding domain (DBD) and a low affinity carboxyl-terminal DBD (1, 2). The RPA2 protein also contains a DBD as well as a phosphorylation site-rich amino terminus that may regulate RPA activity in response to cell cycle phase transitions and DNA damage. Kinases implicated in the phosphorylation of RPA2 include cyclin-dependent kinases, and members of the PI 3-kinase-related kinase superfamily, including DNA-dependent protein kinase (DNA-PK), ataxia-telangiectasia-mutated (ATM), and ATM/Rad3-related (ATR) (3-7). DNA-PK, ATM, and ATR are serine/threonineglutamine (Ser/Thr-Gln)-directed kinases with overlapping substrate specificities that regulate DNA repair, apoptosis, and cell cycle checkpoint responses to genotoxic stimuli (8, 9). At least two Ser/Thr-Gln residues of RPA2 (Thr-21 and Ser-33) are phosphorylated by DNA-PK, and most like...

Ataxia-telangiectasia (A-T) is a syndrome of cancer susceptibility, immune dysfunction, and neurodegeneration that is caused by mutations in the A-T-mutated (ATM) gene. ATM has been implicated as a critical regulator of cellular responses to DNA damage, including the activation of cell cycle checkpoints and induction of apoptosis. Although defective cell cycle-checkpoint regulation and associated genomic instability presumably contribute to cancer susceptibility in A-T, the mechanism of neurodegeneration in A-T is not well understood. In addition, although ATM is required for the induction of the p53 transcriptional program in response to DNA damage, the identities of the relevant transcription factors that mediate ATM-dependent changes in gene expression remain largely undetermined. In this article, we describe a signal transduction pathway linking ATM directly to the Ca 2؉ ͞cAMP response element-binding protein, CREB, a transcription factor that regulates cell growth, homeostasis, and survival. ATM phosphorylated CREB in vitro and in vivo in response to ionizing radiation (IR) and H2O2 on a stress-inducible domain. IR-induced phosphorylation of CREB correlated with a decrease in CREB transactivation potential and reduced interaction between CREB and its transcriptional coactivator, CREB-binding protein (CBP). A CREB mutant containing Ala substitutions at ATM phosphorylation sites displayed enhanced transactivation potential, resistance to inhibition by IR, and increased binding to CBP. We propose that ATM-mediated phosphorylation of CREB in response to DNA damage modulates CREBdependent gene expression and that dysregulation of the ATM-CREB pathway may contribute to neurodegeneration in A-T.A taxia-telangiectasia (A-T) is a recessive genetic syndrome characterized by immune deficiency, cancer susceptibility, and cerebellar degeneration (1). A-T is caused by mutations in ATM, which encodes a protein kinase belonging to the phosphoinositide 3-kinase-related kinase gene superfamily (2). At the cellular level, ATM-deficient cells grow poorly in culture, are genetically unstable and display exquisite sensitivity to ionizing radiation (IR) and radiomimetic drugs (1). A-T cells are also characteristically defective in the G 1 -S, intraS, and G 2 -M cell cycle checkpoints after ␥-irradiation (3), which is believed to contribute to genomic instability and cancer susceptibility.The checkpoint-signaling functions of ATM are achieved by means of the coordinated phosphorylation of polypeptide substrates, including p53, BRCA1, NBS1, and CHK2, which transmit signals to the DNA repair, apoptosis, and cell cycle machinery (3). Far less is known regarding the mechanism of cerebellar degeneration in A-T. Purkinje and granule neurons, which are most severely affected in human A-T, are not grossly abnormal in ATM Ϫ/Ϫ mice (4, 5). However, brains from ATM Ϫ/Ϫ mice display subtle developmental defects and are abnormally resistant to IR-induced apoptosis (6, 7). It has been proposed that neurodegeneration in human A-T is due to the d...

The functionally related ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases are critical regulators of DNA damage responses in mammalian cells. ATM and ATR share highly overlapping substrate specificities and show a strong preference for the phosphorylation of Ser or Thr residues followed by Gln. In this report we used a polyreactive phosphospecific antibody (␣-pDSQ) that recognizes a subset of phosphorylated AspSer-Gln sequences to purify candidate ATM/ATR substrates. This led to the identification of phosphorylation sites in the carboxyl terminus of the minichromosome maintenance protein 3 (MCM3), a component of the hexameric MCM DNA helicase. We show that the ␣-DSQ antibody recognizes tandem DSQ phosphorylation sites (Ser-725 and Ser-732) in the carboxyl terminus of murine MCM3 (mMCM3) and that ATM phosphorylates both sites in vitro. ATM phosphorylated the carboxyl termini of mMCM3 and human MCM3 in vivo and the phosphorylated form of MCM3 retained association with the canonical MCM complex. Although DNA damage did not affect steady-state levels of chromatin-bound MCM3, the ATM-phosphorylated form of MCM3 was preferentially localized to the soluble, nucleoplasmic fraction. This finding suggests that the carboxyl terminus of chromatin-loaded MCM3 may be sequestered from ATM-dependent checkpoint signals. Finally, we show that ATM and ATR jointly contribute to UV lightinduced MCM3 phosphorylation, but that ATM is the predominant UV-activated MCM3 kinase in vivo. The carboxyl-terminal ATM phosphorylation sites are conserved in vertebrate MCM3 orthologs suggesting that this motif may serve important regulatory functions in response to DNA damage. Our findings also suggest that DSQ motifs are common phosphoacceptor motifs for ATM family kinases. The ataxia telangiectasia-mutated (ATM)2 protein kinase is a broad regulator of cellular responses to DNA damage in mammals. Mutations in ATM cause ataxia telangiectasia (A-T), a syndrome characterized by progressive cerebellar degeneration, immune defects, and cancer susceptibility (1). ATM-deficient cells are hypersensitive to ionizing radiation (IR) and radiomimetic agents and exhibit cell cycle checkpoint abnormalities and subtle DNA repair defects. These combined defects in DNA damage signaling and repair are responsible for the 100-fold increased cancer risk associated with A-T, and most likely contribute to the neuropathologic abnormalities associated with this disease.ATM belongs to the extended family of highly conserved phosphoinositide 3-kinase-related kinases (2). Within the mammalian phosphoinositide 3-kinase-related kinase family, ATM is structurally and functionally most closely related to ATR (ATM-Rad3-related) (2). ATM and ATR exhibit similar substrate specificities in vitro, and display a strong preference for the phosphorylation of substrates on Ser/Thr-Gln ((S/T)-Q) motifs. Other (S/T)-Q-directed phosphoinositide 3-kinase-related kinases include the DNA-dependent protein kinase, which mediates DNA repair by non-homologous end ...