SMART MODE:

NORMAL GENOMIC

• Xu X, Nakazawa N, Yanagida M
• Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.
• PLoS One. 2015; 10: 119347-119347
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• Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes) subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo) repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe) mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

• Takada S, Collins ER, Kurahashi K
• The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.
• Mol Biol Cell. 2015; 26: 1811-28
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• DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.

• Paull TT
• Mechanisms of ATM Activation.
• Annu Rev Biochem. 2015; 84: 711-38
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• The ataxia-telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response, and it coordinates checkpoint activation, DNA repair, and metabolic changes in eukaryotic cells in response to DNA double-strand breaks and oxidative stress. Loss of ATM activity in humans results in the pleiotropic neurodegeneration disorder ataxia-telangiectasia. ATM exists in an inactive state in resting cells but can be activated by the Mre11-Rad50-Nbs1 (MRN) complex and other factors at sites of DNA breaks. In addition, oxidation of ATM activates the kinase independently of the MRN complex. This review discusses these mechanisms of activation, as well as the posttranslational modifications that affect this process and the cellular factors that affect the efficiency and specificity of ATM activation and substrate phosphorylation. I highlight functional similarities between the activation mechanisms of ATM, phosphatidylinositol 3-kinases (PI3Ks), and the other PI3K-like kinases, as well as recent structural insights into their regulation.

• Anacker DC, Gautam D, Gillespie KA, Chappell WH, Moody CA
• Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.
• J Virol. 2014; 88: 8528-44
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• Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE: The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication.

• Di Domenico EG et al.
• Multifunctional role of ATM/Tel1 kinase in genome stability: from the DNA damage response to telomere maintenance.
• Biomed Res Int. 2014; 2014: 787404-787404
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• The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development.

• Tobias F et al.
• Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions.
• PLoS One. 2013; 8: 57953-57953
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• The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.

• Berardinelli F, di Masi A, Antoccia A
• NBN Gene Polymorphisms and Cancer Susceptibility: A Systemic Review.
• Curr Genomics. 2013; 14: 425-40
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• The relationship between DNA repair failure and cancer is well established as in the case of rare, high penetrant genes in high cancer risk families. Beside this, in the last two decades, several studies have investigated a possible association between low penetrant polymorphic variants in genes devoted to DNA repair pathways and risk for developing cancer. This relationship would be also supported by the observation that DNA repair processes may be modulated by sequence variants in DNA repair genes, leading to susceptibility to environmental carcinogens. In this framework, the aim of this review is to provide the reader with the state of the art on the association between common genetic variants and cancer risk, limiting the attention to single nucleotide polymorphisms (SNPs) of the NBN gene and providing the various odd ratios (ORs). In this respect, the NBN protein, together with MRE11 and RAD50, is part of the MRN complex which is a central player in the very early steps of sensing and processing of DNA double-strand breaks (DSBs), in telomere maintenance, in cell cycle control, and in genomic integrity in general. So far, many papers were devoted to ascertain possible association between common synonymous and non-synonymous NBN gene polymorphisms and increased cancer risk. However, the results still remain inconsistent and inconclusive also in meta-analysis studies for the most investigated E185Q NBN miscoding variant.

• Gautam D, Bridge E
• The kinase activity of ataxia-telangiectasia mutated interferes with adenovirus E4 mutant DNA replication.
• J Virol. 2013; 87: 8687-96
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• Adenovirus (Ad) mutants that lack early region 4 (E4) are unable to produce the early regulatory proteins that normally inactivate the Mre11/Rad50/Nbs1 (MRN) sensor complex, which is a critical component for the ability of cells to respond to DNA damage. E4 mutant infection therefore activates a DNA damage response, which in turn interferes with a productive viral infection. MRN complex proteins localize to viral DNA replication centers in E4 mutant-infected cells, and this complex is critical for activating the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which phosphorylate numerous substrates important for DNA repair, cell cycle checkpoint activation, and apoptosis. E4 mutant growth defects are substantially rescued in cells lacking an intact MRN complex. We have assessed the role of the downstream ATM and ATR kinases in several MRN-dependent E4 mutant phenotypes. We did not identify a role for either ATM or ATR in "repair" of E4 mutant genomes to form concatemers. ATR was also not observed to contribute to E4 mutant defects in late protein production. In contrast, the kinase activity of ATM was important for preventing efficient E4 mutant DNA replication and late gene expression. Our results suggest that the MRN complex interferes with E4 mutant DNA replication at least in part through its ability to activate ATM.

• Duursma AM, Driscoll R, Elias JE, Cimprich KA
• A role for the MRN complex in ATR activation via TOPBP1 recruitment.
• Mol Cell. 2013; 50: 116-22
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• The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated.

• Sun W et al.
• Crystal structure of the yeast TSC1 core domain and implications for tuberous sclerosis pathological mutations.
• Nat Commun. 2013; 4: 2135-2135
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• Tuberous sclerosis complex is a disease caused by mutations in two tumor-suppressor genes, TSC1 and TSC2. The TSC1 protein, also known as hamartin, has a critical role in controlling mTOR signalling. TSC1 does not bear apparent sequence homology with other proteins. Here we show that the N-terminal half of yeast TSC1 forms a protease-resistant domain, which is evolutionarily conserved. The crystal structure of this yeast TSC1 core domain shows that it contains a pseudo-HEAT repeat fold with its C-terminal end capped by a helical subdomain. This allows us to model the three-dimensional structure of the human TSC1 N-terminal domain (TSC1-NTD), which anchors essentially all pathogenic TSC1 missense mutations found in tuberous sclerosis patients. Interestingly, most pathogenic mutations map inside of the folded TSC1-NTD structure, whereas most non-pathogenic variants are on the structural surface. This indicates that the disruption of the TSC1-NTD globular structure is a major cause of tuberous sclerosis.

• Guerineau M, Kriz Z, Kozakova L, Bednarova K, Janos P, Palecek J
• Analysis of the Nse3/MAGE-binding domain of the Nse4/EID family proteins.
• PLoS One. 2012; 7: 35813-35813
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• BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

• Bleuyard JY, Buisson R, Masson JY, Esashi F
• ChAM, a novel motif that mediates PALB2 intrinsic chromatin binding and facilitates DNA repair.
• EMBO Rep. 2012; 13: 135-41
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• The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the repair of DNA damage by homologous recombination. Here, we report that chromatin-association motif (ChAM), an evolutionarily conserved motif in PALB2, is necessary and sufficient to mediate its chromatin association in both unperturbed and damaged cells. ChAM is distinct from the previously described PALB2 DNA-binding regions. Deletion of ChAM decreases PALB2 and Rad51 accumulation at DNA damage sites and confers cellular hypersensitivity to the genotoxic drug mitomycin C. These results suggest that PALB2 chromatin association via ChAM facilitates PALB2 function in the cellular resistance to DNA damage.

• Yamazaki H, Tarumoto Y, Ishikawa F
• Tel1(ATM) and Rad3(ATR) phosphorylate the telomere protein Ccq1 to recruit telomerase and elongate telomeres in fission yeast.
• Genes Dev. 2012; 26: 241-6
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• In fission yeast, the DNA damage sensor kinases Tel1(ATM) and Rad3(ATR) exist at telomeres and are required for telomere maintenance, but the biological role they play at telomeres is not known. Here we show that the telomere protein Ccq1 is phosphorylated at Thr 93 (threonine residue at amino acid 93) by Tel1(ATM) and Rad3(ATR) both in vitro and in vivo. A ccq1 mutant in which alanine was substituted for Thr 93 failed to recruit telomerase to telomeres and showed gradual shortening of telomeres. These results indicate that the direct phosphorylation of Ccq1 Thr 93 by Tel1 and Rad3 is involved in the recruitment of telomerase to elongate telomeres.

• Bednarski JJ, Sleckman BP
• Lymphocyte development: integration of DNA damage response signaling.
• Adv Immunol. 2012; 116: 175-204
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• Lymphocytes traverse functionally discrete stages as they develop into mature B and T cells. This development is directed by cues from a variety of different cell surface receptors. To complete development, all lymphocytes must express a functional nonautoreactive heterodimeric antigen receptor. The genes that encode antigen receptor chains are assembled through the process of V(D)J recombination, a reaction that proceeds through DNA double-stranded break (DSB) intermediates. These DSBs are generated by the RAG endonuclease in G1-phase developing lymphocytes and activate ataxia-telangiectasia mutated (ATM), the kinase that orchestrates cellular DSB responses. The canonical DNA damage response includes cell cycle arrest, DNA break repair, and apoptosis of cells when DSBs are not repaired. However, recent studies have demonstrated that ATM activation in response to RAG DSBs also regulates a transcriptional program including many genes with no known function in canonical DNA damage responses. Rather, these genes have activities that would be important for lymphocyte development. Here, these findings and the broader concept that signals initiated by physiologic DNA DSBs provide cues that regulate cell type-specific processes and functions are discussed.

• Jang ER, Choi JD, Lee JS
• Acetyltransferase p300 regulates NBS1-mediated DNA damage response.
• FEBS Lett. 2011; 585: 47-52
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• The role of p300 in DNA damage response is unclear. To understand how ATM-dependent phosphorylation of p300 affects its function in response to DNA damage, we present evidence that S106 of p300, which is phosphorylated by ATM, regulates stability of NBS1 and recruitment into damaged DNA, possibly leading to regulation of DNA repair. Non-phosphorylatable p300 (S106A) destabilized NBS1 and decreased NBS1-p300 interaction. The recruitment of NBS1 into damaged DNA was impaired in the presence of S106A. Also, a dominant negative p300 lacking enzymatic activity induced destabilization of NBS1, suggesting that its acetyltransferase is required for NBS1 stability. These results indicate that phosphorylation of p300 can regulate NBS1-mediated DNA damage response, and that these events occur in an acetylation-dependent manner.

• Hudson JJ et al.
• Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID Families.
• PLoS One. 2011; 6: 17270-17270
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• BACKGROUND: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. METHODOLOGY/PRINCIPAL FINDINGS: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. CONCLUSIONS/SIGNIFICANCE: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

• White JS, Yue N, Hu J, Bakkenist CJ
• The ATM kinase signaling induced by the low-energy beta-particles emitted by (33)P is essential for the suppression of chromosome aberrations and is greater than that induced by the energetic beta-particles emitted by (32)P.
• Mutat Res. 2011; 708: 28-36
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• Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cmx5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) beta-particle emitter (32)P-orthophosphate for 30min is approximately 1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) beta-particle emitter (33)P-orthophosphate is approximately 0.18Gy IR. We show that low-energy beta-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic beta-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to approximately 0.36Gy beta-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.

• Dar I et al.
• Investigation of the functional link between ATM and NBS1 in the DNA damage response in the mouse cerebellum.
• J Biol Chem. 2011; 286: 15361-76
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• Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are related genomic instability syndromes characterized by neurological deficits. The NBS1 protein that is defective in NBS is a component of the Mre11/RAD50/NBS1 (MRN) complex, which plays a major role in the early phase of the complex cellular response to double strand breaks (DSBs) in the DNA. Among others, Mre11/RAD50/NBS1 is required for timely activation of the protein kinase ATM (A-T, mutated), which is missing or inactivated in patients with A-T. Understanding the molecular pathology of A-T, primarily its cardinal symptom, cerebellar degeneration, requires investigation of the DSB response in cerebellar neurons, particularly Purkinje cells, which are the first to be lost in A-T patients. Cerebellar cultures derived from mice with different mutations in DNA damage response genes is a useful experimental system to study malfunctioning of the damage response in the nervous system. To clarify the interrelations between murine Nbs1 and Atm, we generated a mouse strain with specific disruption of the Nbs1 gene in the central nervous system on the background of general Atm deficiency (Nbs1-CNS-Delta//Atm(-/-)). This genotype exacerbated several features of both conditions and led to a markedly reduced life span, dramatic decline in the number of cerebellar granule neurons with considerable cerebellar disorganization, abolishment of the white matter, severe reduction in glial cell proliferation, and delayed DSB repair in cerebellar tissue. Combined loss of Nbs1 and Atm in the CNS significantly abrogated the DSB response compared with the single mutation genotypes. Importantly, the data indicate that Atm has cellular roles not regulated by Nbs1 in the murine cerebellum.

• Yeh TL, Lee CY, Amzel LM, Espenshade PJ, Bianchet MA
• The hypoxic regulator of sterol synthesis nro1 is a nuclear import adaptor.
• Structure. 2011; 19: 503-14
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• Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 A resolution shows an all-alpha-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

• Li T, Wang ZQ
• Point mutation at the Nbs1 Threonine 278 site does not affect mouse development, but compromises the Chk2 and Smc1 phosphorylation after DNA damage.
• Mech Ageing Dev. 2011; 132: 382-8
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• NBS1, mutated in Nijmegen breakage syndrome (NBS), senses the DNA double strand breaks (DSBs) and initiates the DNA damage response (DDR) by activating ATM kinase. Meanwhile, NBS1 is phosphorylated by ATM at Serine 278 and Serine 343 and thereby assists the activation of the ATM downstream targets. To study the physiological function of the Nbs1 phosphorylation, we have knocked in a point mutation in the moue genome that results in the replacement of Threonine 278 (equivalent to the human Serine 278) by Alanine. The Nbs1(T278A) knock-in mice develop normally and show no gross defects. The mutation of this phosphorylation site does not affect the proliferation or genomic stability. Ionizing radiation (IR) of primary Nbs1(T278A) MEFs reveals no obvious defects in the Chk2 phosphorylation at 1Gy, but a delayed phosphorylation of Chk2 and Smc1 only at intermediate (4.5Gy) and high (10Gy) doses, respectively. In contrast to Serine 343 mutant, Threonine 278 mutation has no effect on the HU-induced ATR-Chk1 activation. Our study thus shows that Nbs1 phosphorylation at the Threonine 278 is dispensable for mouse development and plays a differential function in assisting the DDR of downstream effectors in vivo, depending on the doses of DNA damage.

• Ivey RG et al.
• Blood-based detection of radiation exposure in humans based on novel phospho-Smc1 ELISA.
• Radiat Res. 2011; 175: 266-81
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• The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1(Ser-957) and phospho-Smc1(Ser-966). Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1(Ser-957) and phospho-Smc1(Ser-966) in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to (131)I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident.

• Xu D et al.
• Rif1 provides a new DNA-binding interface for the Bloom syndrome complex to maintain normal replication.
• EMBO J. 2010; 29: 3140-55
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• BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA-binding domain that resembles the alphaCTD domain of bacterial RNA polymerase alpha; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA-binding interface for the BLM complex to restart stalled replication forks.

• Dodson GE, Limbo O, Nieto D, Russell P
• Phosphorylation-regulated binding of Ctp1 to Nbs1 is critical for repair of DNA double-strand breaks.
• Cell Cycle. 2010; 9: 1516-22
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• Repair of DNA double-strand breaks (DSBs) is critical for cell survival and for maintaining genome stability in eukaryotes. In Schizosaccharomyces pombe, the Mre11-Rad50-Nbs1 (MRN) complex and Ctp1 cooperate to perform the initial steps that process and repair these DNA lesions via homologous recombination (HR). While Ctp1 is recruited to DSBs in an MRN-dependent manner, the specific mechanism of this process remained unclear. We recently found that Ctp1 is phosphorylated on a domain rich in putative Casein kinase 2 (CK2) phosphoacceptor sites that resembles the SDTD repeats of Mdc1. Furthermore, phosphorylation of this motif is required for interaction with the FHA domain of Nbs1 that localizes Ctp1 to DSB sites. Here, we review and discuss these findings, and we present new data that further characterize the cellular consequences of mutating CK2 phosphorylation motifs of Ctp1, including data showing that these sites are critical for meiosis.

• Subramanian L, Nakamura TM
• A kinase-independent role for the Rad3(ATR)-Rad26(ATRIP) complex in recruitment of Tel1(ATM) to telomeres in fission yeast.
• PLoS Genet. 2010; 6: 1000839-1000839
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• ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1(ATM) and Rad3(ATR) recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1(ATM) to telomeres independently of the previously established Nbs1 C-terminal Tel1(ATM) interaction domain. Recruitment of Tel1(ATM) to telomeres in nbs1-c60Delta cells, which lack the C-terminal 60 amino acid Tel1(ATM) interaction domain of Nbs1, was dependent on Rad3(ATR)-Rad26(ATRIP), but the kinase domain of Rad3(ATR) was dispensable. Thus, our results establish that the Rad3(ATR)-Rad26(ATRIP) complex contributes to the recruitment of Tel1(ATM) independently of Rad3(ATR) kinase activity, by a mechanism redundant with the Tel1(ATM) interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3(ATR)-Rad26(ATRIP) to telomeres. In response to replication stress, mammalian ATR-ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR-ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity.

• Wrzeszczynski KO, Rost B
• Cell cycle kinases predicted from conserved biophysical properties.
• Proteins. 2009; 74: 655-68
• Display abstract

• Machine-learning techniques can classify functionally related proteins where homology-transfer as well as sequence and structure motifs fail. Here, we present a method that aimed at complementing homology-transfer in the identification of cell cycle control kinases from sequence alone. First, we identified functionally significant residues in cell cycle proteins through their high sequence conservation and biophysical properties. We then incorporated these residues and their features into support vector machines (SVM) to identify new kinases and more specifically to differentiate cell cycle kinases from other kinases and other proteins. As expected, the most informative residues tend to be highly conserved and tend to localize in the ATP binding regions of the kinases. Another observation confirmed that ATP binding regions are typically not found on the surface but in partially buried sites, and that this fact is correctly captured by accessibility predictions. Using these highly conserved, semi-buried residues and their biophysical properties, we could distinguish cell cycle S/T kinases from other kinase families at levels around 70-80% accuracy and 62-81% coverage. An application to the entire human proteome predicted at least 97 human proteins with limited previous annotations to be candidates for cell cycle kinases.

• Kinoshita E, van der Linden E, Sanchez H, Wyman C
• RAD50, an SMC family member with multiple roles in DNA break repair: how does ATP affect function?
• Chromosome Res. 2009; 17: 277-88
• Display abstract

• The protein complex including Mre11, Rad50, and Nbs1 (MRN) functions in DNA double-strand break repair to recognize and process DNA ends as well as signal for cell cycle arrest. Amino acid sequence similarity and overall architecture make Rad50 a member of the structural maintenance of chromosome (SMC) protein family. Like SMC proteins, Rad50 function depends on ATP binding and hydrolysis. All current evidence indicates that ATP binding and hydrolysis cause architectural rearrangements in SMC protein complexes that are important for their functions in organizing DNA. In the case of the MRN complex, the functional significance of ATP binding and hydrolysis are not yet defined. Here we review the data on the ATP-dependent activities of MRN and their possible mechanistic significance. We present some speculation on the role of ATP for function of the MRN complex based on the similarities and differences in the molecular architecture of the Rad50-containing complexes and the SMC complexes condensin and cohesin.

• Lloyd J et al.
• A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage.
• Cell. 2009; 139: 100-11
• Display abstract

• The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.

• van der Linden E, Sanchez H, Kinoshita E, Kanaar R, Wyman C
• RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.
• Nucleic Acids Res. 2009; 37: 1580-8
• Display abstract

• The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.

• Cimprich KA, Cortez D
• ATR: an essential regulator of genome integrity.
• Nat Rev Mol Cell Biol. 2008; 9: 616-27
• Display abstract

• Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.

• Subramanian L, Moser BA, Nakamura TM
• Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast.
• Mol Cell Biol. 2008; 28: 1443-55
• Display abstract

• Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Delta survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Delta trt1Delta cells are able to stably maintain telomeres. Thus, taz1Delta trt1Delta cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Delta trt1Delta cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Delta trt1Delta cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.

• Xie H, Wise SS, Wise JP Sr
• Deficient repair of particulate hexavalent chromium-induced DNA double strand breaks leads to neoplastic transformation.
• Mutat Res. 2008; 649: 230-8
• Display abstract

• Hexavalent chromium (Cr(VI)) is a potent respiratory toxicant and carcinogen. The most carcinogenic forms of Cr(VI) are the particulate salts such as lead chromate, which deposit and persist in the respiratory tract after inhalation. We demonstrate here that particulate chromate induces DNA double strand breaks in human lung cells with 0.1, 0.5, and 1 microg/cm(2) lead chromate inducing 1.5, 2, and 5 relative increases in the percent of DNA in the comet tail, respectively. These lesions are repaired within 24 h and require Mre11 expression for their repair. Particulate chromate also caused Mre11 to co-localize with gamma-H2A.X and ATM. Failure to repair these breaks with Mre11-induced neoplastic transformation including loss of cell contact inhibition and anchorage-independent growth. A 5-day exposure to lead chromate induced loss of cell contact inhibition in a concentration-dependent manner with 0, 0.1, 0.5, and 1 microg/cm(2) lead chromate inducing 1, 78, and 103 foci in 20 dishes, respectively. These data indicate that Mre11 is critical to repairing particulate Cr(VI)-induced double strand breaks and preventing Cr(VI)-induced neoplastic transformation.

• Kinner A, Wu W, Staudt C, Iliakis G
• Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.
• Nucleic Acids Res. 2008; 36: 5678-94
• Display abstract

• DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

• Mordes DA, Cortez D
• Activation of ATR and related PIKKs.
• Cell Cycle. 2008; 7: 2809-12
• Display abstract

• The DNA damage response kinase ATR is an essential regulator of genome integrity. TopBP1 functions as a general activator of ATR. We have recently shown that TopBP1 activates ATR through its regulatory subunit ATRIP and a PIKK regulatory domain (PRD) located adjacent to its kinase domain. This mechanism of ATR activation is conserved in the S. cerevisiae ortholog Mec1. ATR is a member of the PIKK family of protein kinases that includes ATM, DNA-PKcs, mTOR and SMG1. The PRD regulates the kinase activity of other PIKKs and may serve as a site of interaction between these kinase and their respective activators. Activation of ATR by TopBP1 is maximal at low substrate concentrations and declines exponentially as substrate concentration increases. These data are consistent with a model in which TopBP1 acts to alter the conformation of ATR-ATRIP to increase the ability of ATR to bind substrates. A further understanding of the mechanism of ATR activation will likely provide insights into the regulation of related PIKKs.

• Rahal EA, Henricksen LA, Li Y, Turchi JJ, Pawelczak KS, Dixon K
• ATM mediates repression of DNA end-degradation in an ATP-dependent manner.
• DNA Repair (Amst). 2008; 7: 464-75
• Display abstract

• Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.

• Seidel JJ, Anderson CM, Blackburn EH
• A novel Tel1/ATM N-terminal motif, TAN, is essential for telomere length maintenance and a DNA damage response.
• Mol Cell Biol. 2008; 28: 5736-46
• Display abstract

• Tel1/ATM, a conserved phosphatidylinositol 3-kinase-related kinase (PIKK), acts in the response to DNA damage and regulates telomere maintenance. PIKK family members share an extended N-terminal region of low sequence homology. Sequence alignment of the N terminus of Tel1/ATM orthologs revealed a conserved, novel motif we term TAN (for Tel1/ATM N-terminal motif). Point mutations in conserved residues of the TAN motif resulted in telomere shortening, and its deletion caused the same short telomere phenotype as complete deletion of Tel1 did. Overexpressing Tel1 TAN mutants did not rescue telomere shortening. The TAN motif was also essential for the function of Tel1 in the response to DNA damage, as TAN-deleted Tel1 was indistinguishable from the complete lack of Tel1 in causing reduced viability and signaling through Rad53 upon DNA damage. Strikingly, TAN deletion reduced recruitment of Tel1 to a double-strand DNA break. Together, these results define a conserved sequence motif within an otherwise poorly defined region of the Tel1/ATM kinase family proteins that is essential for normal Tel1 function in Saccharomyces cerevisiae.

• Daniel JA, Pellegrini M, Lee JH, Paull TT, Feigenbaum L, Nussenzweig A
• Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo.
• J Cell Biol. 2008; 183: 777-83
• Display abstract

• Cellular responses to both physiological and pathological DNA double-strand breaks are initiated through activation of the evolutionarily conserved ataxia telangiectasia mutated (ATM) kinase. Upon DNA damage, an activation mechanism involving autophosphorylation has been reported to allow ATM to phosphorylate downstream targets important for cell cycle checkpoints and DNA repair. In humans, serine residues 367, 1893, and 1981 have been shown to be autophosphorylation sites that are individually required for ATM activation. To test the physiological importance of these sites, we generated a transgenic mouse model in which all three conserved ATM serine autophosphorylation sites (S367/1899/1987) have been replaced with alanine. In this study, we show that ATM-dependent responses at both cellular and organismal levels are functional in mice that express a triple serine mutant form of ATM as their sole ATM species. These results lend further support to the notion that ATM autophosphorylation correlates with the DNA damage-induced activation of the kinase but is not required for ATM function in vivo.

• Stracker TH, Couto SS, Cordon-Cardo C, Matos T, Petrini JH
• Chk2 suppresses the oncogenic potential of DNA replication-associated DNA damage.
• Mol Cell. 2008; 31: 21-32
• Display abstract

• The Mre11 complex (Mre11, Rad50, and Nbs1) and Chk2 have been implicated in the DNA-damage response, an inducible process required for the suppression of malignancy. The Mre11 complex is predominantly required for repair and checkpoint activation in S phase, whereas Chk2 governs apoptosis. We examined the relationship between the Mre11 complex and Chk2 in the DNA-damage response via the establishment of Nbs1(DeltaB/DeltaB) Chk2(-/-) and Mre11(ATLD1/ATLD1) Chk2(-/-) mice. Chk2 deficiency did not modify the checkpoint defects or chromosomal instability of Mre11 complex mutants; however, the double-mutant mice exhibited synergistic defects in DNA-damage-induced p53 regulation and apoptosis. Nbs1(DeltaB/DeltaB) Chk2(-/-) and Mre11(ATLD1/ATLD1) Chk2(-/-) mice were also predisposed to tumors. In contrast, DNA-PKcs-deficient mice, in which G1-specific chromosome breaks are present, did not exhibit synergy with Chk2(-/-) mutants. These data suggest that Chk2 suppresses the oncogenic potential of DNA damage arising during S and G2 phases of the cell cycle.

• Iijima K et al.
• NBS1 regulates a novel apoptotic pathway through Bax activation.
• DNA Repair (Amst). 2008; 7: 1705-16
• Display abstract

• DNA damage induced apoptosis, along with precise DNA damage repair, is a critical cellular function, and both of these functions are necessary for cancer prevention. The NBS1 protein is known to be a key regulator of DNA damage repair. It acts by forming a complex with Rad50/Mre11 and by activating ATM. We show here that NBS1 regulates a novel p53 independent apoptotic pathway in response to DNA damage. DNA damage induced apoptosis was significantly reduced in NBS1 deficient cells regardless of their p53 status. Experiments using a series of cell lines expressing mutant NBS1 proteins revealed that NBS1 is able to regulate the activation of Bax and Caspase-3 without the FHA, Mre11-binding, or the ATM-interacting domains, whereas the phosphorylation sites of NBS1 were essential for Bax activation. Expression of apoptosis-related transcription factors such as E2F1 and their downstream pro-apoptotic factors were not related to this apoptosis induction. Interestingly, NBS1 regulates a novel Bax activation pathway by disrupting the Ku70-Bax complex which is required for activation of the mitochondrial apoptotic pathway. This dissociation of the Ku70-Bax complex can be mediated by acetylation of Ku70, and NBS1 can function in this process through a protein-protein interaction with Ku70. Thus, NBS1 is a key protein involved in the prevention of carcinogenesis, not only through the precise repair of damaged DNA by homologous recombination (HR) but also by its role in the elimination of inappropriately repaired cells.

• You Z, Bailis JM, Johnson SA, Dilworth SM, Hunter T
• Rapid activation of ATM on DNA flanking double-strand breaks.
• Nat Cell Biol. 2007; 9: 1311-8
• Display abstract

• The tumour-suppressor gene ATM, mutations in which cause the human genetic disease ataxia telangiectasia (A-T), encodes a key protein kinase that controls the cellular response to DNA double-strand breaks (DSBs). DNA DSBs caused by ionizing radiation or chemicals result in rapid ATM autophosphorylation, leading to checkpoint activation and phosphorylation of substrates that regulate cell-cycle progression, DNA repair, transcription and cell death. However, the precise mechanism by which damaged DNA induces ATM and checkpoint activation remains unclear. Here, we demonstrate that linear DNA fragments added to Xenopus egg extracts mimic DSBs in genomic DNA and provide a platform for ATM autophosphorylation and activation. ATM autophosphorylation and phosphorylation of its substrate NBS1 are dependent on DNA fragment length and the concentration of DNA ends. The minimal DNA length required for efficient ATM autophosphorylation is approximately 200 base pairs, with cooperative autophosphorylation induced by DNA fragments of at least 400 base pairs. Importantly, full ATM activation requires it to bind to DNA regions flanking DSB ends. These findings reveal a direct role for DNA flanking DSB ends in ATM activation.

• Cariveau MJ, Tang X, Cui XL, Xu B
• Characterization of an NBS1 C-terminal peptide that can inhibit ataxia telangiectasia mutated (ATM)-mediated DNA damage responses and enhance radiosensitivity.
• Mol Pharmacol. 2007; 72: 320-6
• Display abstract

• ATM and NBS1, mutation of which lead to the human autosomal recessive diseases ataxia telangiectasia and Nijmegen breakage syndrome (NBS), respectively, are essential elements in the cellular response to DNA damage induced by ionizing radiation (IR). ATM is a member of the phosphatidylinositol 3-kinase family and is activated by IR in an NBS1-dependent manner. The extreme C terminus of NBS1 contains an evolutionarily conserved sequence motif that is critical for binding to and activation of ATM after IR. ATM phosphorylates a series of targets to initiate cell cycle arrest and promote cell survival in response to DNA damage. Therefore, targeting the NBS1-ATM interaction may lead to a novel approach for specific ATM inhibition and radiosensitization. We developed small peptides containing the conserved C-terminal sequence of NBS1 to investigate whether these peptides can interfere with the DNA damage pathway. We found that wild-type NBS1 inhibitory peptides (wtNIP) can abrogate NBS1-ATM association in the presence or absence of IR. We also found that cells exposed to wtNIP displayed a significant reduction in radiation-induced gamma-H2AX and NBS1 focus formation compared with cells treated with control peptides, demonstrating that wtNIP possesses a strong inhibitory effect on ATM. The inhibitory effect of wtNIP also leads to a significant decrease in clonogenic survival in response to IR. Furthermore, wtNIP does not radiosensitize cells with defective ATM, suggesting a specific inhibition of ATM. Together, these data provide a proof of principle for the use of NBS1 C-terminal small peptides as specific ATM inhibitors and radiosensitizers.

• Goldberg JM et al.
• The dictyostelium kinome--analysis of the protein kinases from a simple model organism.
• PLoS Genet. 2006; 2: 38-38
• Display abstract

• Dictyostelium discoideum is a widely studied model organism with both unicellular and multicellular forms in its developmental cycle. The Dictyostelium genome encodes 285 predicted protein kinases, similar to the count of the much more advanced Drosophila. It contains members of most kinase classes shared by fungi and metazoans, as well as many previously thought to be metazoan specific, indicating that they have been secondarily lost from the fungal lineage. This includes the entire tyrosine kinase-like (TKL) group, which is expanded in Dictyostelium and includes several novel receptor kinases. Dictyostelium lacks tyrosine kinase group kinases, and most tyrosine phosphorylation appears to be mediated by TKL kinases. About half of Dictyostelium kinases occur in subfamilies not present in yeast or metazoa, suggesting that protein kinases have played key roles in the adaptation of Dictyostelium to its habitat. This study offers insights into kinase evolution and provides a focus for signaling analysis in this system.

• Takemura H et al.
• Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks.
• J Biol Chem. 2006; 281: 30814-23
• Display abstract

• The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.

• Jiang X, Sun Y, Chen S, Roy K, Price BD
• The FATC domains of PIKK proteins are functionally equivalent and participate in the Tip60-dependent activation of DNA-PKcs and ATM.
• J Biol Chem. 2006; 281: 15741-6
• Display abstract

• Members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including the ATM, DNA-PKcs, Atr, and Trrap proteins, function in signal transduction pathways that activate the DNA damage response. PIKK proteins contain a conserved C-terminal FAT/kinase domain/FATC domain structure. The FATC domain of ATM mediates the interaction between ATM and Tip60, a histone acetyltransferase that regulates activation of ATM. Here, we examined whether the FATC domains of DNA-PKcs, Atr, and Trrap were also able to interact with Tip60. Deletion of the FATC domain of ATM blocked the interaction between ATM and Tip60 and suppressed the activation of ATM kinase activity by DNA damage. Replacement of the FATC domain of ATM with the FATC domains of DNA-PKcs, Atr, or Trrap restored the activation of ATM and its association with Tip60. These results indicate that the FATC domains of DNA-PKcs, Atr, Trrap, and ATM are functionally equivalent. Immunoprecipitation experiments demonstrated that Tip60 is constitutively associated with DNA-PKcs and that the histone acetyltransferase activity associated with DNA-PKcs is up-regulated by DNA damage. When Tip60 expression was suppressed by small interfering RNA, the activation of DNA-PKcs (measured by autophosphorylation of DNA-PKcs at serine 2056 and threonine 2609) was inhibited, demonstrating a key role for Tip60 in the activation of DNA-PKcs by DNA damage. The conserved FATC domain of PIKK proteins may therefore function as a binding domain for the Tip60 histone acetyltransferase. Further, the ability of Tip60 to regulate the activation of both ATM and DNA-PKcs in response to DNA damage demonstrates that Tip60 is a key component of the DNA damage-signaling network.

• Dupre A, Boyer-Chatenet L, Gautier J
• Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex.
• Nat Struct Mol Biol. 2006; 13: 451-7
• Display abstract

• DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. We propose that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome.

• De Souza CP, Hashmi SB, Horn KP, Osmani SA
• A point mutation in the Aspergillus nidulans sonBNup98 nuclear pore complex gene causes conditional DNA damage sensitivity.
• Genetics. 2006; 174: 1881-93
• Display abstract

• The nuclear pore complex (NPC) is embedded in the nuclear envelope where it mediates transport between the cytoplasm and nucleus and helps to organize nuclear architecture. We previously isolated sonB1, a mutation encoding a single amino acid substitution within the Aspergillus nidulans SONBnNup98 NPC protein (nucleoporin). Here we demonstrate that this mutation causes marked DNA damage sensitivity at 42 degrees . Although SONBnNup98 has roles in the G2 transition, we demonstrate that the G2 DNA damage checkpoint is functional in the sonB1 mutant at 42 degrees . The MRN complex is composed of MRE11, RAD50, and NBS1 and functions in checkpoint signaling, DNA repair, and telomere maintenance. At 42 degrees we find that the DNA damage response defect of sonB1 mutants causes synthetic lethality when combined with mutations in scaANBS1, the A. nidulans homolog of NBS1. We provide evidence that this synthetic lethality is independent of MRN cell cycle checkpoint functions or MREAMRE11-mediated DNA repair functions. We also demonstrate that the single A. nidulans histone H2A gene contains the C-terminal SQE motif of histone H2AX isoforms and that this motif is required for the DNA damage response. We propose that the sonB1 nucleoporin mutation causes a defect in a novel part of the DNA damage response.

• Farah JA, Cromie G, Steiner WW, Smith GR
• A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe.
• Genetics. 2005; 169: 1261-74
• Display abstract

• DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.

• Lee JH, Paull TT
• ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
• Science. 2005; 308: 551-4
• Display abstract

• The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.

• Andrews CA, Clarke DJ
• MRX (Mre11/Rad50/Xrs2) mutants reveal dual intra-S-phase checkpoint systems in budding yeast.
• Cell Cycle. 2005; 4: 1073-7
• Display abstract

• The intra-S-phase checkpoint is a signaling pathway that induces slow DNA replication in the presence of DNA damage. In humans, defects in this checkpoint pathway might account for phenotypes seen in autosomal recessive diseases including ataxia telangiectasia-like disorder and Nijmegen breakage syndrome, where MRN complex components,Mre11 and Nbs1, are mutated. Here we provide evidence that the equivalent budding yeast complex, MRX (Mre11/Rad50/Xrs2), is not required for the intra-S-phase checkpoint in response to DNA alkylation damage, but is required in the presence of double-stranded DNA breaks. These data indicate, at least in budding yeast, that alternate pathways enforce replication slowing depending on the particular DNA lesion.

• Zhong H, Bryson A, Eckersdorff M, Ferguson DO
• Rad50 depletion impacts upon ATR-dependent DNA damage responses.
• Hum Mol Genet. 2005; 14: 2685-93
• Display abstract

• The Mre11/Rad50/NBS1 (MRN) complex is mutated in inherited genomic instability syndromes featuring cancer predisposition, mental retardation and immunodeficiency. It functions both in DNA double-strand break repair and in controlling the ataxia telangiectasia mutated (ATM) kinase during the response to these lesions. Patients inheriting homozygosity for an NBS1 hypomorphic allele display reduced phosphorylation of signaling factors such as Chk1, but not of chromatin-associated factor H2AX, after stresses that activate the ATM-related kinase, ATR. Therefore, we tested whether MRN has a global controlling role over the ATR kinase through the study of MRN deficiencies generated via RNA interference. We show for the first time that MRN is required for ATR-dependent phosphorylation of structural maintenance of chromosomes 1 (Smc1), which acts within chromatin to ensure sister chromatid cohesion and to effect several DNA damage responses. We have uncovered novel phenotypes caused by MRN deficiency that support a functional link between this complex, ATR and Smc1, including hypersensitivity to UV exposure, a defective UV responsive intra-S phase checkpoint and a specific pattern of genomic instability. In addition, certain ATR-dependent responses do not require MRN. These studies demonstrate that there is indeed a controlling role for MRN over the ATR kinase and have established that the downstream events under this control are broad, including both chromatin-associated and diffuse signaling factors, but may not be universal. These studies contribute to our understanding of the central role that MRN plays in damage detection and signaling, which serve to maintain genomic stability and resist neoplastic transformation.

• McSherry TD, Mueller PR
• Xenopus Cds1 is regulated by DNA-dependent protein kinase and ATR during the cell cycle checkpoint response to double-stranded DNA ends.
• Mol Cell Biol. 2004; 24: 9968-85
• Display abstract

• The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.

• Yoo HY, Shevchenko A, Shevchenko A, Dunphy WG
• Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses.
• J Biol Chem. 2004; 279: 53353-64
• Display abstract

• In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. In this study, we describe the isolation of a cDNA encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, we have identified the essential replication protein Mcm2 as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 underwent phosphorylation at Ser(92) in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involved both ATM and ATR, but the relative contribution of each kinase depended upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylated Mcm2 directly at Ser(92) in cell-free kinase assays. Immunodepletion of both ATM and ATR abrogated the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts.

• Zhang Z, Hu W, Cano L, Lee TD, Chen DJ, Chen Y
• Solution structure of the C-terminal domain of Ku80 suggests important sites for protein-protein interactions.
• Structure. 2004; 12: 495-502
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• The solution structure of Ku80 CTD from residue 566 to 732 has been solved in order to gain insights into the mechanisms of its interactions with other proteins. The structure reveals a topology similar to several common scaffolds for protein-protein interactions, in the absence of significant sequence similarity to these proteins. Conserved surface amino acid residues are clustered on two main surface areas, which are likely involved in mediating interactions between Ku80 and other proteins. The Ku70/Ku80 heterodimer has been shown to be involved in at least three processes, nonhomologous end joining, transcription, and telomere maintenance, and thus it needs to interact with different proteins involved in these different processes. The three-dimensional structure of the Ku80 C-terminal domain and the availability of NMR chemical shift assignments provide a basis for further investigation of the interactions between Ku80 and other proteins in these Ku-dependent cellular functions.

• Nakada D, Hirano Y, Sugimoto K
• Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.
• Mol Cell Biol. 2004; 24: 10016-25
• Display abstract

• The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

• Perry J, Kleckner N
• The ATRs, ATMs, and TORs are giant HEAT repeat proteins.
• Cell. 2003; 112: 151-5

• Hopfner KP, Tainer JA
• Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures.
• Curr Opin Struct Biol. 2003; 13: 249-55
• Display abstract

• ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines for diverse biological pathways. ABC ATPase domains act not only in ABC transporters but also in DNA mismatch, nucleotide excision and double-strand break repair enzymes, as well as in chromosome segregation. Atomic-resolution crystal structures suggest molecular mechanisms for ABC ATPases and reveal surprisingly significant mechanistic and architectural conservation. This emerging unified structural biochemistry provides general medical and biological insights into how ABC proteins function as chemo-mechanical devices. ATP binding by the signature and Q-loop motifs drives the conformations of substrate-specific domains to accomplish diverse functions in transmembrane transport and DNA repair.

• Carson CT, Schwartz RA, Stracker TH, Lilley CE, Lee DV, Weitzman MD
• The Mre11 complex is required for ATM activation and the G2/M checkpoint.
• EMBO J. 2003; 22: 6610-20
• Display abstract

• The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.

• Hodson JA, Bailis JM, Forsburg SL
• Efficient labeling of fission yeast Schizosaccharomyces pombe with thymidine and BUdR.
• Nucleic Acids Res. 2003; 31: 134-134
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• In this paper we report the construction of a Schizosaccharomyces pombe strain that facilitates analysis of replicating DNA. The strain co-expresses the Herpes simplex virus thymidine kinase gene (hsv-tk) and a human equilibrative nucleoside transporter (hENT1). The double integrant efficiently incorporates 3H-thymidine into nuclear DNA as monitored by scintillation counting. These strains also incorporate the thymidine analog Bromodeoxy uridine (BUdR) into newly replicated DNA, which can be detected by immunofluorescence and flow cytometry. This strain provides a valuable tool for direct study of DNA replication in S.pombe.

• Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W
• Histone H2A variants H2AX and H2AZ.
• Curr Opin Genet Dev. 2002; 12: 162-9
• Display abstract

• Two of the nucleosomal histone families, H3 and H2A, have highly conserved variants with specialized functions. Recent studies have begun to elucidate the roles of two of the H2A variants, H2AX and H2AZ. H2AX is phosphorylated on a serine four residues from the carboxyl terminus in response to the introduction of DNA double-strand breaks, whether these breaks are a result of environmental insult, metabolic mistake, or programmed process. H2AZ appears to alter nucleosome stability, is partially redundant with nucleosome remodeling complexes, and is involved in transcriptional control.

• Williams BR, Mirzoeva OK, Morgan WF, Lin J, Dunnick W, Petrini JH
• A murine model of Nijmegen breakage syndrome.
• Curr Biol. 2002; 12: 648-53
• Display abstract

• Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.

• Nyberg KA, Michelson RJ, Putnam CW, Weinert TA
• Toward maintaining the genome: DNA damage and replication checkpoints.
• Annu Rev Genet. 2002; 36: 617-56
• Display abstract

• DNA checkpoints play a significant role in cancer pathology, perhaps most notably in maintaining genome stability. This review summarizes the genetic and molecular mechanisms of checkpoint activation in response to DNA damage. The major checkpoint proteins common to all eukaryotes are identified and discussed, together with how the checkpoint proteins interact to induce arrest within each cell cycle phase. Also discussed are the molecular signals that activate checkpoint responses, including single-strand DNA, double-strand breaks, and aberrant replication forks. We address the connection between checkpoint proteins and damage repair mechanisms, how cells recover from an arrest response, and additional roles that checkpoint proteins play in DNA metabolism. Finally, the connection between checkpoint gene mutation and genomic instability is considered.

• Nakamura TM, Moser BA, Russell P
• Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres.
• Genetics. 2002; 161: 1437-52
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• Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.

• Kobayashi J et al.
• NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain.
• Curr Biol. 2002; 12: 1846-51
• Display abstract

• DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.

• Wakayama T, Kondo T, Ando S, Matsumoto K, Sugimoto K
• Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae.
• Mol Cell Biol. 2001; 21: 755-64
• Display abstract

• In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Delta mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Delta cells, as in mec1Delta cells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1Delta mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Delta mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

• Hopfner KP et al.
• Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
• Cell. 2000; 101: 789-800
• Display abstract

• To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

• Chook YM, Blobel G
• Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp.
• Nature. 1999; 399: 230-7
• Display abstract

• Transport factors in the karyopherin-beta (also called importin-beta) family mediate the movement of macromolecules in nuclear-cytoplasmic transport pathways. Karyopherin-beta2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran x GTP binds karyopherin-beta2 and dissociates the substrate. Here we present the 3.0 A structure of the karyopherin-beta2-Ran x GppNHp complex where GppNHp is a non-hydrolysable GTP analogue. Karyopherin-beta2 contains eighteen HEAT repeats arranged into two continuous orthogonal arches. Ran is clamped in the amino-terminal arch and substrate-binding activity is mapped to the carboxy-terminal arch. A large loop in HEAT repeat 7 spans both arches. Interactions of the loop with Ran and the C-terminal arch implicate it in GTPase-mediated dissociation of the import-substrate. Ran x GppNHp in the complex shows extensive structural rearrangement, compared to Ran GDP, in regions contacting karyopherin-beta2. This provides a structural basis for the specificity of the karyopherin-beta family for the GTP-bound state of Ran, as well as a rationale for interactions of the karyopherin-Ran complex with the regulatory proteins ranGAP, ranGEF and ranBP1.

• Groves MR, Hanlon N, Turowski P, Hemmings BA, Barford D
• The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.
• Cell. 1999; 96: 99-110
• Display abstract

• The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge.

• Haber JE
• The many interfaces of Mre11.
• Cell. 1998; 95: 583-6

• Lavin MF, Shiloh Y
• The genetic defect in ataxia-telangiectasia.
• Annu Rev Immunol. 1997; 15: 177-202
• Display abstract

• The autosomal recessive human disorder ataxia-telangiectasia (A-T) was first described as a separate disease entity 40 years ago. It is a multisystem disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, radiosensitivity, predisposition to lymphoid malignancies and immunodeficiency, with defects in both cellular and humoral immunity. The pleiotropic nature of the clinical and cellular phenotype suggests that the gene product involved is important in maintaining stability of the genome but also plays a more general role in signal transduction. The chromosomal instability and radiosensitivity so characteristic of this disease appear to be related to defective activation of cell cycle checkpoints. Greater insight into the nature of the defect in A-T has been provided by the recent identification, by positional cloning, of the responsible gene, ATM. The ATM gene is related to a family of genes involved in cellular responses to DNA damage and/or cell cycle control. These genes encode large proteins containing a phosphatidylinositol 3-kinase domain, some of which have protein kinase activity. The mutations causing A-T completely inactivate or eliminate the ATM protein. This protein has been detected and localized to different subcellular compartments.

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