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DNA damage response is finely tuned, with several pathways including those for DNA repair, chromatin remodeling and cell cycle checkpoint, although most studies to date have focused on single pathways. Genetic diseases characterized by genome instability have provided novel insights into the underlying mechanisms of DNA damage response. NBS1, a protein responsible for the radiation-sensitive autosomal recessive disorder Nijmegen breakage syndrome, is one of the first factors to accumulate at sites of DNA double-strand breaks (DSBs). NBS1 binds to at least five key proteins, including ATM, RPA, MRE11, RAD18 and RNF20, in the conserved regions within a limited span of the C terminus, functioning in the regulation of chromatin remodeling, cell cycle checkpoint and DNA repair in response to DSBs. In this article, we reviewed the functions of these binding proteins and their comprehensive association with NBS1., (© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Low-dose radiation risks remain unclear owing to a lack of sufficient studies. We previously reported that low-dose, long-term fractionated radiation (FR) with 0.01 or 0.05 Gy/fraction for 31 d inflicts oxidative stress in human fibroblasts due to excess levels of mitochondrial reactive oxygen species (ROS). To identify the small effects of low-dose radiation, we investigated how mitochondria respond to low-dose radiation in radiosensitive human ataxia telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)1-deficient cell lines compared with corresponding cell lines expressing ATM and NBS1. Consistent with previous results in normal fibroblasts, low-dose, long-term FR increased mitochondrial mass and caused accumulation of mitochondrial ROS in ATM- and NBS1-complemented cell lines. Excess mitochondrial ROS resulted in mitochondrial damage that was in turn recognized by Parkin, leading to mitochondrial autophagy (mitophagy). In contrast, ATM- and NBS1-deficient cells showed defective induction of mitophagy after low-dose, long-term FR, leading to accumulation of abnormal mitochondria; this was determined by mitochondrial fragmentation and decreased mitochondrial membrane potential. Consequently, apoptosis was induced in ATM- and NBS1-deficient cells after low-dose, long-term FR. Antioxidant N-acetyl-L-cysteine was effective as a radioprotective agent against mitochondrial damage induced by low-dose, long-term FR among all cell lines, including radiosensitive cell lines. In conclusion, we demonstrated that mitochondria are target organelles of low-dose radiation. Mitochondrial response influences radiation sensitivity in human cells. Our findings provide new insights into cancer risk estimation associated with low-dose radiation exposure.

Nijmegen breakage syndrome (NBS) is a recessive genetic disorder characterized by increased sensitivity to ionizing radiation (IR) and a high frequency of malignancies. NBS1, a product of the mutated gene in NBS, contains several protein interaction domains in the N-terminus and C-terminus. The C-terminus of NBS1 is essential for interactions with MRE11, a homologous recombination repair nuclease, and ATM, a key player in signal transduction after the generation of DNA double-strand breaks (DSBs), which is induced by IR. Moreover, NBS1 regulates chromatin remodeling during DSB repair by histone H2B ubiquitination through binding to RNF20 at the C-terminus. Thus, NBS1 is considered as the first protein to be recruited to DSB sites, wherein it acts as a sensor or mediator of DSB damage responses. In addition to DSB response, we showed that NBS1 initiates Polη-dependent translesion DNA synthesis by recruiting RAD18 through its binding at the NBS1 C-terminus after UV exposure, and it also functions after the generation of interstrand crosslink DNA damage. Thus, NBS1 has multifunctional roles in response to DNA damage from a variety of genotoxic agents, including IR.

Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5' to 3' strand resection (DSB resection), with nucleases generating the 3' single-strand DNA (3'ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.

Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy., (© The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Centrosomes are important cytoplasmic organelles involved in chromosome segregation, defects in which can result in aneuploidy, and contribute to tumorigenesis. It is known that DNA damage causes the supernumerary centrosomes by a mechanism in which centrosomes continue to duplicate during cell cycle arrest at checkpoints. We show here that ionizing radiation induces the overduplication of centrosomes in a dose-dependent manner, and that the level of overduplication is pronounced in BRCA1- and NBS1-deficient cells, even though their checkpoint control is abrogated. Conversely, marginal increases in overduplication were observed in Ku70- and DNA-PKcs-deficient cells, which are intact in checkpoint control. The frequency of radiation-induced overduplication of centrosomes might be associated with DNA repair, as it was decreased with reduced cell killing after protracted exposures to radiation. As a result, when the frequency of radiation-induced centrosome overduplication was plotted against radiation-induced cell killing, similar curves were seen for both protracted and acute exposures in wild-type cells, Ku70-deficient, and DNA-PKcs-deficient cells, indicating a common mechanism for centrosome overduplication. However, the absence of either BRCA1 or NBS1 enhanced radiation-induced overduplication frequencies by 2-4-fold on the basis of the same cell killing. These results suggest that radiation-induced centrosome overduplication is regulated by at least two mechanisms: a checkpoint-dependent pathway involved in wild-type cells, Ku70-deficient and DNA-PKcs-deficient cells; and a checkpoint-independent pathway as observed in BRCA1-deficient and NBS1-deficient cells., (© 2010 Japanese Cancer Association.)

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of poleta and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Phosphorylated histone H2AX (gamma-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with gamma-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether gamma-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a gamma-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-gamma-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, gamma-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.

Nijmegen breakage syndrome is characterized by genomic instability and a predisposition for lymphoma and solid tumors. Nijmegen breakage syndrome 1 (NBS1), the protein which is mutated in these patients, functions in association with BRCA1 and ATR as part of the cellular response to DNA double-strand breaks. We show here that NBS1 forms foci at the centrosomes via an interaction with gamma-tubulin. Down-regulation of NBS1 by small interfering RNA induces supernumerary centrosomes, and this was confirmed with experiments using Nbs1 knockout mouse cells; the introduction of wild-type NBS1 (wt-NBS1) cDNA into these knockout mouse cells reduced the number of supernumerary centrosomes to normal levels. This phenotype in NBS1-deficient cells is caused by both centrosome duplication and impaired separation of centrioles, which have been observed in BRCA1-inhibited cells. In fact, supernumerary centrosomes were observed in Brca1 knockout mouse cells, and the frequency was not affected by NBS1 down-regulation, suggesting that NBS1 maintains centrosomes via a common pathway with BRCA1. This is consistent with findings that NBS1 physically interacts with BRCA1 at the centrosomes and is required for BRCA1-mediated ubiquitination of gamma-tubulin. Moreover, the ubiquitination of gamma-tubulin is compromised by either ATR depletion or an NBS1 mutation in the ATR interacting (FHA) domain, which is essential for ATR activation. These results suggest that, although centrosomes lack DNA, the NBS1/ATR/BRCA1 repair machinery affects centrosome behavior, and this might be a crucial role in the prevention of malignances.

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.

NBS1 forms a multimetric complex with MRE11/RAD50, which acts as the sensor of DNA double-strand breaks (DSBs). The mechanisms controlling the expression of NBS1 remain largely unknown. Here we show that NBS1 is transcribed as both a wild-type and an alternatively spliced form exhibiting a premature stop codon in an alternative 50-bp exon in intron 2. Although the wild-type transcript predominates in most tissues, the spliced transcript is abundant in resting peripheral blood mononuclear cells (PBMCs). Levels of the spliced form of NBS1 decreased rapidly after irradiation as levels of the wild-type NBS1 transcript increased, resulting in increased levels of NBS1 protein. Both mitogenic stimulation and methyl methanesulfonate treatment also altered the splicing pattern of NBS1. Resting PBMCs, which predominantly express spliced NBS1, were more susceptible to radiation than mitogen-stimulated cells, which showed predominant expression of the wild-type transcript. Since the alternatively spliced NBS1 gene likely did not produce protein, this alternative splicing seems to be associated with the control of NBS1 protein. Thus alternative splicing of the NBS1 gene may be associated with the regulation of NBS1 in response to DSBs, DNA alkylation damage, and mitogenic response.

TopBP1 is involved in DNA replication and DNA damage checkpoint. Recent studies have demonstrated that TopBP1 is a direct positive effecter of ATR. However, it is not known how TopBP1 recognizes damaged DNA. Here, we show that TopBP1 formed nuclear foci after exposure to ionizing radiation, but such TopBP1 foci were abolished in Nijmegen breakage syndrome cells. We also show that TopBP1 physically associated with NBS1 in vivo. These results suggested that NBS1 might regulate TopBP1 recruitment to the sites of DNA damage. TopBP1-depleted cells showed hypersensitivity to Mitomycin C and ionizing radiation, an increased frequency of sister-chromatid exchange level, and a reduced frequency of DNA double-strand break induced homologous recombination repair. Together, these results suggested that TopBP1 might be a mediator of DNA damage signaling from NBS1 to ATR and promote homologous recombination repair.

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.

The WRN protein is mutated in the chromosomally unstable Werner syndrome (WS) and the Nbs1 protein is mutated in Nijmegen breakage syndrome (NBS). The Nbs1 protein is an integral component of the M/R/N complex. Although WRN is known to interact with this complex in response to gamma-irradiation, the mechanism of action is unclear. Here, we show that WRN co-localizes and associates with gamma H2AX, a marker protein of DNA double strand breaks (DSBs), after cellular exposure to gamma-irradiation. While the DNA damage-inducible Nbs1 foci formation is normal in WS cells, WRN focus formation is defective in NBS cells. Consistent with this, gamma H2AX colocalizes with Nbs1 in WS cells but not with WRN in NBS cells. The defective WRN-gamma H2AX association in NBS cells can be complemented with wild-type Nbs1, but not with an Nbs1 S343A point mutant that lacks an ATM phosphorylation site. WRN associates with H2AX in a manner dependent upon the M/R/N complex. Our results suggest a novel pathway in which Nbs1 may recruit WRN to the site of DNA DSBs in an ATM-dependent manner.

Artemis plays a crucial role in the hairpin-opening step of antigen receptor VDJ gene recombination in the presence of catalytic subunit of deoxyribonucleic acid (DNA)-dependent protein kinase (DNA-PKcs). A defect in Artemis causes human radiosensitive-severe combined immunodeficiency. Cells from Artemis-deficient patients and mice display increased chromosomal instability, but the precise function of this factor in the response to DNA damage remains to be elucidate. In this study, we show that Artemis is hyperphosphorylated in an Ataxia-telangiectasia-mutated (ATM)- and Nijmegen breakage syndrome 1 (Nbs1)-dependent manner in response to ionizing radiation (IR), and that S645 is an SQ/TQ site that contributes to retarded mobility of Artemis upon IR. The hyperphosphorylation of Artemis is markedly reduced in ATM- and Nbs1-null cells. Reintroduction of wild-type ATM or Nbs1 reconstituted Artemis hyperphosphorylation in ATM- or Nbs1-deficient cells, respectively. In support of this functional link, hyperphosphorylated Artemis was found to physically associate with the Mre11/Rad50/Nbs1 complex in an ATM-dependent manner in response to IR-induced DNA double strand breaks (DSB). Since deficiency of either DNA-Pkcs or ATM leads to defective repair of IR-induced DSB, our finding places Artemis at the signaling crossroads downstream of DNA-PKcs and ATM in IR-induced DSB repair.

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

NBS1 is the key regulator of the RAD50/MRE11/NBS1 (R/M/N) protein complex, a sensor and mediator for cellular DNA damage response. NBS1 potentiates the enzymatic activity of MRE11 and directs the R/M/N complex to sites of DNA damage, where it forms nuclear foci by interacting with phosphorylated H2AX. The R/M/N complex also activates the ATM kinase, which is a major kinase involved in the activation of DNA damage signal pathways. The ATM requires the R/M/N complex for its own activation following DNA damage, and for conformational change to develop a high affinity for target proteins. In addition, association of NBS1 with PML, the promyelocytic leukemia protein, is required to form nuclear bodies, which have various functions depending on their location and composition. These nuclear bodies function not only in response to DNA damage, but are also involved in telomere maintenance when they are located on telomeres. In this review, we describe the role of NBS1 in the maintenance of genetic stability through the activation of cell-cycle checkpoints, DNA repair, and protein relocation.

Nijmegen breakage syndrome is a recessive genetic disorder, characterized by elevated sensitivity to ionizing radiation, chromosome instability and high frequency of malignancies. Since cellular features partly overlap with those of ataxia-telangiectasia (A-T), NBS was long considered an A-T clinical variant. NBS1, the product of the gene underlying the disease, contains three functional regions: the forkhead-associated (FHA) domain and BRCA1 C-terminus (BRCT) domain at the N-terminus, several SQ motifs (consensus phosphorylation sites by ATM and ATR kinases) at a central region and MRE11-binding region at the C-terminus. NBS1 forms a multimeric complex with hMRE11/hRAD50 nuclease at the C-terminus and recruits or retains them at the vicinity of sites of DNA damage by direct binding to histone H2AX, which is phosphorylated by ATM in response to DNA damage. The combination of the FHA/BRCT domains has a crucial role for the binding of NBS1 to H2AX. Thereafter, the NBS1 complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates, while it also might be involved in suppression of inter-chromosomal recombination even for V(D)J recombination. These processes collaborate with cell cycle checkpoints to facilitate DNA repair, while defects of these checkpoints in NBS cells are partial in nature. A possible explanation for these moderate defects are the redundancy of multiple checkpoint regulations in vertebrates, or the modulator role of NBS1, in which NBS1 amplifies ATM activation by accumulation of the MRN complex at damaged sites. This molecular link of NBS1 to ATM may explain the phenotypic similarity of NBS to A-T.

The Werner syndrome and the Nijmegen breakage syndrome are recessive genetic disorders that show increased genomic instability, cancer predisposition, hypersensitivity to mitomycin C and gamma-irradiation, shortened telomeres, and cell cycle defects. The protein mutated in the premature aging disease known as the Werner syndrome is designated WRN and is a member of the RecQ helicase family. The Nbs1 protein is mutated in Nijmegen breakage syndrome individuals and is part of the mammalian Mre11 complex together with the Mre11 and Rad50 proteins. Here, we show that WRN associates with the Mre11 complex via binding to Nbs1 in vitro and in vivo. In response to gamma-irradiation or mitomycin C, WRN leaves the nucleoli and co-localizes with the Mre11 complex in the nucleoplasm. We detect an increased association between WRN and the Mre11 complex after cellular exposure to gamma-irradiation. Small interfering RNA and complementation experiments demonstrated convergence of WRN and Nbs1 in response to gamma-irradiation or mitomycin C. Nbs1 is required for the Mre11 complex promotion of WRN helicase activity. Taken together, these results demonstrate a functional link between the two genetic diseases with partially overlapping phenotypes in a pathway that responds to DNA double strand breaks and interstrand cross-links.

The isolation of the NBS1 gene revealed the molecular mechanisms of DSB repair. In response to DNA damage, histone H2AX in the vicinity of DSBs is phosphorylated by ATM. NBS1 then targets the MRE11/RAD50 complex to the sites of DSBs through interaction of the FHA/BRCT domain with gamma-H2AX. NBS1 complex binds to damaged-DNA directly, and HR repair is initiated. To collaborate DSB repair, ATM also regulates cell cycle checkpoints at G1, G2, and intra-S phases via phosphorylation of SMC, CHK2 and FANCD2. The phosphorylation of these proteins require NBS1 complex. Thus, NBS1 has at least two important roles in genome maintenance, as a DNA repair protein in HR pathway and as a signal modifier in intra-S phase checkpoints. NBS1 is also known to be involved in maintenance of telomeres, which have DSB-like structures and defects here can cause telomeric fusion. Therefore, NBS1 should be a multifunctional protein for the maintenance of genomic integrity. Further studies on NBS1 will provide insights into the mechanisms of DNA damage response and the network of these factors involved in genomic stability.

DNA double-strand breaks represent the most potentially serious damage to a genome and hence, at least two pathways of DNA repair have evolved; namely, homologous recombination repair and non-homologous end joining. Defects in both rejoining processes result in genomic instability including chromosome rearrangements, LOH and gene mutations, which may lead to development of malignancies. Nijmegen breakage syndrome is a recessive genetic disorder, characterized by elevated sensitivity to ionizing radiation that induces double-strand breaks, and high frequency of malignancies. NBS1, the product of the gene underlying the disease, forms a multimeric complex with hMRE11/hRAD50 nuclease and recruits them to the vicinity of sites of DNA damage by direct binding to phosphorylated histone H2AX. The combination of the highly-conserved NBS1 forkhead associated domain and BRCA1 C-terminus domain has a crucial role for recognition of damaged sites. Thereafter, the NBS1-complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates. This process collaborates with cell-cycle checkpoints at S and G2 phase to facilitate DNA repair. NBS1 is also associated with telomere maintenance and DNA replication. Based on recent knowledge regarding NBS1, we propose here a two-step binding mechanism for damage recognition by repair proteins, and describe the molecular links to factors for genome stability.

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.

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.