Abstract: Maintenance of genome stability is essential for avoiding the passage to neoplasia. The DNA-damage response--a cornerstone of genome stability--occurs by a swift transduction of the DNA-damage signal to many cellular pathways. A prime example is the cellular response to DNA double-strand breaks, which activate the ATM protein kinase that, in turn, modulates numerous signalling pathways. ATM mutations lead to the cancer-predisposing genetic disorder ataxia-telangiectasia (A-T). Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.

Abstract: The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.

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.

Abstract: NFBD1/MDC1 (mediator of DNA damage checkpoint 1) is a nuclear factor with an amino-terminal FHA (forkhead-associated) domain and a tandem repeat of BRCT (breast cancer susceptibility gene-1 carboxyl terminus) domains. We have previously shown that NFBD1 is an early participant in DNA damage signaling pathways and that ionizing radiation-induced nuclear foci (IRIF) of NFBD1 colocalize with several DNA checkpoint signaling and repair factors. We report here that NFBD1 physically associates with ATM, p53, components of the MRE11-RAD50-NBS1 (MRN) complex, and γ-H2AX. An overexpressed FHA domain-containing fragment of NFBD1 binds to endogenous NFBD1 and components of the MRN complex, but not to γ-H2AX. This fragment interferes with IRIF formation by endogenous NFBD1, MRE11, or NBS1. A BRCT domain-containing fragment of NFBD1 binds to γ-H2AX and 53BP1, but not to components of the MRN complex, and abolishes IRIF formation by NFBD1, MRE11, NBS1, 53BP1, CHK2 phospho-T68, γ-H2AX, and possible ATM/ATR substrates re...

Abstract: Nijmegen breakage syndrome (NBS) is an autosomal genetic disease demonstrating a variety of phenotypic abnormalities, including premature aging, increased cancer incidence, chromosome instability, and sensitivity to ionizing radiation. The gene involved in NBS, NBS1, is part of the MRE11/RAD50/NBS1 (MRN) complex that also includes MRE11 and RAD50, which is involved in DNA repair and cell cycle regulation in response to DNA damage. The MRN complex is also involved in telomere maintenance, as demonstrated by the shortened telomeres in NBS primary human fibroblasts and the association of NBS1 with the telomere-binding protein TRF2. To learn more about how a deficiency in telomere maintenance might contribute to chromosome instability in NBS, we have investigated the stability of telomeres in two telomerase-positive human tumor cell clones, BNmt-On and BNmt-Off, expressing an inducible NBS1S278A/S343A gene containing mutations at serines 278 and 343 phosphorylated by ATM. The results demonstrate an increased rate of telomere loss in both clones following expression of NBS1S278A/S343A. The absence of detectable changes in average telomere length suggests that NBS1-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. The recombination events associated with telomere loss were found to be similar to those shown previously to result in breakage/fusion/bridge cycles, suggesting that telomere loss can contribute to chromosome instability in NBS1-deficient cells. Telomere loss showed no correlation with radiosensitivity or radioresistant DNA synthesis, demonstrating that NBS1S278A/S343A promotes telomere loss through a separate pathway from these other phenotypes associated with NBS.

Abstract: Objective Enhanced DNA double-strand break (DSB) repair could be a primary cause for development of resistance in tumor cells to cisplatin, which induces crosslinks and DNA DSBs. A protein complex consisting of hMre11, hRad50, and Nbs1 (MRN) has been identified as a critical component in repair of DNA DSBs. The present study investigates whether the expression of a truncated form of Nbs1 interrupts the function of the MRN complex and therefore enhances cisplatin-induced DNA damage and cytotoxicity in human head and neck squamous cell carcinoma (HNSCC). Methods and measures Two human HNSCC cell lines, JHU006 and JHU029, were used. A dominant negative recombinant adenovirus expressing domains of Nbs1 was constructed. Adenovirus-mediated mutant Nbs1 (Ad-Nbs1) gene transfer was performed with replication-defective virus (DL312) and no treatment as controls. Transgene expression and cell viability were evaluated in transfected cells. Neutral comet assay was performed and the “tail moment,” the product of the amount of DNA in the tail and the distance of tail migration, was analyzed for evaluating DNA DSB damage at 24, 48, and 72 hours. Results Transgene expression of mutant Nbs1 was confirmed by Western blotting. Ad-Nbs1 gene transfer significantly increased cisplatin-induced cytotoxicity as shown by stunting of 6-day growth curves. Neutral comet analysis revealed that the mean tail moment, indicative of DNA damage, was significantly elevated in cells treated with combined cisplatin and Ad-Nbs1 compared to cisplatin alone in both cell lines. Conclusions Expression of mutant Nbs1 significantly increases cisplatin-induced DNA DSBs and cytotoxicity. The increase in double-strand DNA damage corresponds to the level of cytotoxicity in the different treatment groups and suggests that tumor chemosensitization occurs through augmentation of DNA DSBs. Clinical significance Alteration of DNA repair may provide a novel approach to enhancing sensitivity of HNSCC to chemotherapy. Our study supports the potential application of Ad-Nbs1 in combination with cisplatin for treatment of advanced and metastatic HNSCC.

Abstract: The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1+ homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Δ mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to γ-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases.

Abstract: The MRE11–RAD50–NBS1 (MRN) protein complex has been linked to many DNA metabolic events that involve DNA double-stranded breaks (DSBs). In vertebrate cells, all three components are encoded by essential genes, and hypomorphic mutations in any of the human genes can result in genome-instability syndromes. MRN is one of the first factors to be localized to the DNA lesion, where it might initially have a structural role by tethering together, and therefore stabilizing, broken chromosomes. This suggests that MRN could function as a lesion-specific sensor. As well as binding to DNA, MRN has other roles in both the processing and assembly of large macromolecular complexes (known as foci) that facilitate efficient DSB responses. Recently, a novel mediator protein, mediator of DNA damage checkpoint protein 1 (MDC1), was shown to co-immunoprecipitate with the MRN complex and regulate MRE11 foci formation. However, whether the initial recruitment of MRN to DSBs requires MDC1 is unclear. Here, we focus on recent developments in MRN research and propose a model for how DSBs are sensed and the cellular responses to them are mediated.