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The study explored proteomics to better understand the relationship between type 2 diabetes (T2DM) and hypertension (HT) in Thai adults, using shotgun proteomics and bioinformatics analysis. Plasma samples were taken from 61 subjects: 14 healthy subjects (mean age = 40.85 ± 7.12), 13 with T2DM (mean age = 57.38 ± 6.03), 16 with HT (mean age = 66.87 ± 10.09), and 18 with coexisting T2DM/HT (mean age = 58.22 ± 10.65). Proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein–protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 11.5. We identified six unique proteins in T2DM patients, including translationally controlled 1 (TPT1) and nibrin (NBN), which are associated with the DNA damage response. In HT patients, seven unique proteins were identified, among them long-chain fatty acid-CoA ligase (ASCL), which functions in the stimulation of triacylglycerol and cholesterol synthesis, and NADPH oxidase activator 1 (NOXA1), which is involved in high blood pressure via angiotensin II-induced reactive oxygen species (ROS)-generating systems. In coexisting T2DM/HT patients, six unique proteins were identified, of which two—microtubule-associated protein 1A (MAP1A)—might be involved in dementia via RhoB-p53 and diacylglycerol kinase beta (DGKB), associated with lipid metabolism. This study identified new candidate proteins that are possibly involved in the pathology of these diseases. [ABSTRACT FROM AUTHOR]

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double‐stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome‐wide, the in vivo off‐target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2‐induced DNA breaks in mouse pre‐B cells on a genome‐wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2‐dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2‐induced DNA DSBs at GA‐ and CA‐repeats and GC‐rich motifs. These findings provide further insights into RAG1/2 off‐target activity. The ability of RAG1/2 to introduce DSBs on the non‐Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes. [ABSTRACT FROM AUTHOR]

In the meiotic prophase, programmed SPO11‐linked DNA double‐strand breaks (DSBs) are repaired by homologous recombination (HR). The MRE11‐RAD50‐NBS1 (MRN) complex is essential for initiating DNA end resection, the first step of HR. However, residual DNA end resection still occurs in Nbs1 knockout (KO) spermatocytes for unknown reasons. Here, we show that DNA end resection is completely abolished in Mre11 KO spermatocytes. In addition, Mre11 KO, but not Nbs1 KO, undifferentiated spermatogonia are rapidly exhausted due to DSB accumulation, proliferation defects, and elevated apoptosis. Cellular studies reveal that a small amount of MRE11 retained in the nucleus of Nbs1 KO cells likely underlies the differences between Mre11 and Nbs1 KO cells. Taken together, our study not only demonstrates an irreplaceable role of the MRE11 in DNA end resection at SPO11‐linked DSBs but also unveils a unique function of MRE11 in maintaining the long‐term viability of undifferentiated spermatogonia. [ABSTRACT FROM AUTHOR]

Disruption of mechanisms that maintain genome stability is an essential factor of tumor progression. Accordingly, predisposition to the development of neoplasms is often associated with germline mutations in genes involved in DNA damage detection and repair. At the same time, impairment of DNA repair systems may be a predictor of antitumor treatment efficacy while overexpression of genes involved in DNA repair is a frequent event in various types of malignancies that can lead to development of tumor cells’ resistance to chemo- and radiotherapy. NBN (nibrin) gene encodes the subunit of the MRN complex which acts as a sensor of double-strand DNA breaks and participates in their repair by homologous recombination. Germline variants in NBN which are associated with increased risk of tumor development are generally represented by frameshift mutations that lead to the synthesis of truncated protein as well as by nonsense and some missense mutations which occur in functionally significant domains. These germline mutations result in partial loss of nibrin function and in increased frequency of spontaneous and induced chromosomal aberrations in the cells of the carriers. On the contrary, amplification of NBN locus is a predominant type of somatic mutations affecting this gene, which indicates a dual role of NBN protein in tumor progression. The results of several studies demonstrate the influence of NBN expression level and its mutational status on anti-tumor drug resistance in particular types of tumor cells and on the survival rate of patients. These data indicate that an in-depth study of different variants and their functional significance is necessary since NBN status may be essential for the choice of treatment tactics for some types of tumors.

Background: DNA damage response (DDR) and repair are vital for safeguarding genetic information and ensuring the survival and accurate transmission of genetic material. DNA damage, such as DNA double-strand breaks (DSBs), triggers a response where sensor proteins recognize DSBs. Information is transmitted to kinases, initiating a sequence resulting in the activation of the DNA damage response and recruitment of other DDR and repair proteins to the DSB site in a highly orderly sequence. Research has traditionally focused on individual protein functions and their order, with limited quantitative analysis, prompting this study's attempt at absolute quantification of DNA damage response and repair proteins and capturing changes in protein chromatin affinity after DNA damage through biochemical fractionation methods. Results: To assess the intracellular levels of proteins involved in DDR and repair, multiple proteins associated with different functions were quantified in EPC2-hTERT cells. H2AX had the highest intracellular abundance (1.93 × 106 molecules/cell). The components of the MRN complex were present at the comparable levels: 6.89 × 104 (MRE11), 2.17 × 104 (RAD50), and 2.35 × 104 (NBS1) molecules/cell. MDC1 was present at 1.27 × 104 molecules/cell. The intracellular levels of ATM and ATR kinases were relatively low: 555 and 4860 molecules/cell, respectively. The levels of cellular proteins involved in NHEJ (53BP1: 3.03 × 104; XRCC5: 2.62 × 104; XRCC6: 5.05 × 105 molecules/cell) were more than an order of magnitude higher than that involved in HR (RAD51: 2500 molecules/cell). Furthermore, we analyzed the dynamics of MDC1 and γH2AX proteins in response to DNA damage induced by the unstable agent neocarzinostatin (NCS). Using cell biochemical fractionation, cells were collected and analyzed at different time points after NCS exposure. Results showed that γH2AX in chromatin fraction peaked at 1 h post-exposure and gradually decreased, while MDC1 translocated from the isotonic to the hypertonic fraction, peaking at 1 hour as well. The study suggests increased MDC1 affinity for chromatin through binding to γH2AX induced by DNA damage. The γH2AX-bound MDC1 (in the hypertonic fraction) to γH2AX ratio at 1 h post-exposure was 1:56.4, with lower MDC1 levels which may attributed to competition with other proteins. Conclusions: The approach provided quantitative insights into protein dynamics in DNA damage response. [ABSTRACT FROM AUTHOR]

The primary goal of this systematic analysis is to determine the predictive significance of proliferative markers in surgical margins of patients with oral squamous cell carcinoma (OSCC). A thorough literature search was done on databases like MEDLINE/Pub-Med, Cochrane and Scopus libraries for similar studies until December 2022. All the relevant original research studies (retrospective and prospective) published in the literature assessing the predictive value of proliferative markers in surgical margins in OSCC were included. Seventeen studies with 1159 patients were included. The research included here used p53, p44/p42, proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR), Ki-67, Bcl2, Nibrin, AgNORs, Cyclin B1, Cornulin, ISG 15antibodies, MCM3 in OSCC. Four studies were done on oral premalignant lesions and OSCC. Among these studies, Ki-67 was the most accurate, followed by p53 (75%) and AgNORs, while PCNA had the least accuracy. To minimize the risk of bias panel of antibodies was suggested in most studies. For interobserver variability, analysis of variance and Chi-square test were used in most studies. The chance of recurrence rate was calculated using a log-rank test and a Kaplan–Meier curve. The significance of proliferative markers in surgical margins of OSCC has been emphasized in the present review. Future research should focus on selecting antibodies, preferably a panel, with a large sample size and extended follow-up. [ABSTRACT FROM AUTHOR]

Aim: To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids. Methods: The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was generated by the stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of doxorubicin (Dox) for 5 months. Half-inhibitory concentrations (IC50) for Dox, vinblastine (Vin), and paclitaxel (PTX) were calculated by a colorimetric MTS-based assay. Crystal violet staining was used to assess cellular viability, whereas the proliferation capacities of cancer cells were monitored in real-time by the i-Celligence system. Expression of apoptotic markers (e.g., cleaved PARP and caspase-3), DNA repair proteins (e.g., ATM, DNA-PK, Nbs1, Rad51, MSH2, etc.), and certain ABC transporters (P-glycoprotein, MRP1, ABCG2, etc.) was assessed by western blotting and real-time PCR. Flow cytometry was used to examine the fluorescence intensity of Dox and ABC-transporter substrates (e.g., Calcein AM and CMFDA) and to assess their excretion to define the activity of specific ABC-transporters. To confirm OS resistance to Dox in vivo, xenograft experiments were performed. Results: An OS subline generated by a stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of Dox resulted in an increase in the IC50 for Dox, Vin, and PTX (~6-, 4-, and 30-fold, respectively). The acquisition of chemoresistance in vitro was also evidenced by the lack of apoptotic markers (e.g., cleaved PARP and caspase-3) in resistant OS cells treated with the chemotherapeutic agents indicated above. The development of the multidrug resistance (MDR) phenotype in this OS subline was due to the overexpression of ABCB1 (i.e., P-glycoprotein) and ABCC1 (i.e., multidrug resistance protein-1, MRP-1), which was evidenced on both mRNA and protein levels. Due to increased expression of MDR-related proteins, resistant OS exhibited an excessive efflux of Dox. Moreover, decreased accumulation of calcein AM, a well-known fluorescent substrate for both ABCB1 and ABCC1, was observed for resistant OS cells compared to their parental SaOS-2 cell line. Importantly, tariquidar and cyclosporin, well-known ABC inhibitors, retained the intensity of Dox-induced fluorescence in resistant SAOS-2 cells. Furthermore, in addition to the increased efflux of the chemotherapeutic agents from Dox-resistant OS cells, we found higher expression of several DNA repair proteins (e.g., Rad51 recombinase, Mre11, and Nbs1, activated forms of ATM, DNA-PK, Chk1, and Chk2, etc.), contributing to the chemoresistance due to the excessive DNA repair. Lastly, the in vivo study indicated that Dox has no impact on the SaOS-2 Dox-R xenograft tumor growth in a nude mouse model. Conclusions: An acquired resistance of OS to the chemotherapeutic agents might be due to the several mechanisms undergoing simultaneously on the single-cell level. This reveals the complexity of the mechanisms involved in the secondary resistance of OS to chemotherapies. [ABSTRACT FROM AUTHOR]

PHRF1 is an E3 ligase that promotes TGF-β signaling by ubiquitinating a homeodomain repressor TG-interacting factor (TGIF). The suppression of PHRF1 activity by PML-RARα facilitates the progression of acute promyelocytic leukemia (APL). PHRF1 also contributes to non-homologous end-joining in response to DNA damage by linking H3K36me3 and NBS1 with DNA repair machinery. However, its role in class switch recombination (CSR) is not well understood. In this study, we report the importance of PHRF1 in IgA switching in CH12F3-2A cells and CD19-Cre mice. Our studies revealed that Crispr-Cas9 mediated PHRF1 knockout and shRNA-silenced CH12F3-2A cells reduced IgA production, as well as decreased the amounts of PARP1, NELF-A, and NELF-D. The introduction of PARP1 could partially restore IgA production in PHRF1 knockout cells. Intriguingly, IgA, as well as IgG1, IgG2a, and IgG3, switchings were not significantly decreased in PHRF1 deficient splenic B lymphocytes isolated from CD19-Cre mice. The levels of PARP1 and NELF-D were not decreased in PHRF1-depleted primary splenic B cells. Overall, our findings suggest that PHRF1 may modulate IgA switching in CH12F3-2A cells. [ABSTRACT FROM AUTHOR]

Aims: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder caused by hypomorphic mutations of NBS1. NBS1 is a member of the MRE11‐RAD50‐NBS1 (MRN) complex that binds to DNA double‐strand breaks and activates the DNA damage response (DDR). Nbs1 inactivation in neural progenitor cells leads to microcephaly and premature death. Interestingly, p53 homozygous deletion rescues the NBS1‐deficient phenotype allowing long‐term survival. The objective of this work was to determine whether simultaneous inactivation of Nbs1 and p53 in neural progenitors triggered brain tumorigenesis and if so in which category this tumour could be classified. Methods: We generated a mouse model with simultaneous genetic inactivation of Nbs1 and p53 in embryonic neural stem cells and analysed the arising tumours with in‐depth molecular analyses including immunohistochemistry, array comparative genomic hybridisation (aCGH), whole exome‐sequencing and RNA‐sequencing. Results: NBS1/P53‐deficient mice develop high‐grade gliomas (HGG) arising in the olfactory bulbs and in the cortex along the rostral migratory stream. In‐depth molecular analyses using immunohistochemistry, aCGH, whole exome‐sequencing and RNA‐sequencing revealed striking similarities to paediatric human HGG with shared features with radiation‐induced gliomas (RIGs). Conclusions: Our findings show that concomitant inactivation of Nbs1 and p53 in mice promotes HGG with RIG features. This model could be useful for preclinical studies to improve the prognosis of these deadly tumours, but it also highlights the singularity of NBS1 among the other DNA damage response proteins in the aetiology of brain tumours. [ABSTRACT FROM AUTHOR]

DNA double strand breaks (DSBs) are a deleterious form of DNA damage. Densely ionising alpha radiation predominantly induces complex DSBs and sparsely ionising gamma radiation—simple DSBs. We have shown that alphas and gammas, when applied simultaneously, interact in producing a higher DNA damage response (DDR) than predicted by additivity. The mechanisms of the interaction remain obscure. The present study aimed at testing whether the sequence of exposure to alphas and gammas has an impact on the DDR, visualised by live NBS1-GFP (green fluorescent protein) focus dynamics in U2OS cells. Focus formation, decay, intensity and mobility were analysed up to 5 h post exposure. Focus frequencies directly after sequential alpha → gamma and gamma → alpha exposure were similar to gamma alone, but gamma → alpha foci quickly declined below the expected values. Focus intensities and areas following alpha alone and alpha → gamma were larger than after gamma alone and gamma → alpha. Focus movement was most strongly attenuated by alpha → gamma. Overall, sequential alpha → gamma exposure induced the strongest change in characteristics and dynamics of NBS1-GFP foci. Possible explanation is that activation of the DDR is stronger when alpha-induced DNA damage precedes gamma-induced DNA damage. [ABSTRACT FROM AUTHOR]

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

Background: Oral cancer is alarming disease in the developing countries like India. DNA repair capacity may affect by genetic polymorphisms in DNA repair genes and thus may cause to cancer. XRCC3 involves in homologous recombination repair pathway and repair DNA damage and crosslinks while, NBS1 participate in repair of double strand DNA break and starts the cell-cycle checkpoint signaling. Aims and Objectives: This study was to conducted to find the association of XRCC3, NBS1 polymorphisms with oral disease. Materials and Methods: This study included 230 patients of precancerous oral lesions (Leukoplakia 70, Oral Sub mucous fibrosis 90, Lichen Planus 70), 72 oral cancer and 300 healthy control samples and genotyping was done by PCR-RFLP methods. Chi-square test was used for calculation of genotype and allele frequencies. Odds ratio and P values were calculated with Epi-Info programme (http://wwwn.cdc.gov/epiinfo/). Results: TT genotype of XRCC3 was associated with high risk of precancerous lesions and oral cancerous lesions (P value=0.0001, OR=9.68, 95% CI=2.82-33.21; and P value=0.0001, OR=13.10, 95% CI=3.38-50.73 respectively). We did not observe any interactions of XRCC3 polymorphism with demographic parameters in influencing the risk of oral diseases. Variant allele genotypes (CG, GG) of NBS1 (C>G) polymorphism showed protective association with Oral submucous fibrosis (OSMF), lichen planus as well as oral cancer (OR=0.31, OR=0.01; OR=0.39, OR=0.03; OR=0.43, OR=0.31 respectively). Particularly, tobacco chewer with CG & GG genotypes were at decrease risk of oral diseases (P value=0.02, OR=0.32, 95% CI=0.12-0.80). Compared to CC/CC combined genotype CG/CC, CG/CT, GG/CC and CG/CT genotypes decreased the risk of oral disease (OR=0.05, 0.47, 0.26 & 0.14 respectively). Conclusion: This study concludes that SNP in XRCC3, NBS1 affects susceptibility to oral disease. [ABSTRACT FROM AUTHOR]

Multidrug resistance-associated proteins are ABC C-family exporters. They are crucial in pharmacology as they transport various substrates across membranes. However, the role of the degenerate nucleotide-binding site (NBS) remains unclear likewise the interplay with the surrounding lipid environment. Here, we propose a dynamic and structural overview of MRP1 from ca. 110 μs molecular dynamics simulations. ATP binding to NBS1 is likely maintained along several transport cycles. Asymmetric NBD behaviour is ensured by lower signal transduction from NBD1 to the rest of the protein owing to the absence of ball-and-socket conformation between NBD1 and coupling helices. Even though surrounding lipids play an active role in the allosteric communication between the substrate-binding pocket and NBDs, our results suggest that lipid composition has a limited impact, mostly by affecting transport kinetics. We believe that our work can be extended to other degenerate NBS ABC proteins and provide hints for deciphering mechanistic differences among ABC transporters. Molecular dynamics simulations of multidrug resistance protein 1 (MRP1) with varying membrane composition suggest that the composition has limited impact on the structures, but rather affects the conformational transitions and kinetics of substrate transport. [ABSTRACT FROM AUTHOR]

The MRE11--RAD50--NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs. [ABSTRACT FROM AUTHOR]

Aims: Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB‐prone animal model. Materials and methods: We generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)‐dependent MB‐prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1‐depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA‐Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs. Results: We found that monoallelic Nbn deletion increases SmoA1‐dependent MB incidence. In addition to a defective DDR, Nbn+/− GCPs show increased clonogenicity compared to Nbn+/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full NbnKO impairs MB development both in SmoA1 mice and in an SHH‐driven tumour allograft. Conclusions: Our study indicates that Nbn is haploinsufficient for SHH‐MB development whereas full NbnKO is epistatic on SHH‐driven MB development, thus revealing a gene dosage‐dependent effect of Nbn inactivation on SHH‐MB development. [ABSTRACT FROM AUTHOR]

Nijmegen breakage syndrome 1 (NBS1) protein is a core member of the MRE11-RAD50-NBS1 (MRN) complex that plays a crucial role in DNA damage sensing and repair in plants. Here we report that NBS1 from moss Physcomitrium patens reduces oxidative damage by lowering the cellular ROS in addition to its known role in oxidative DNA damage recovery. Real-time transcript analysis showed up-regulation of the PpNBS1 transcript under different stress conditions. Bacterial cells showed better cell survivability upon over-expressing PpNBS1 protein as compared to untransformed cells. Likewise, overexpression of PpNBS1 in tobacco plants provides improved protection against oxidative damage and exhibited a lesser amount of ROS upon exposure to oxidative stress. Moreover, PpNBS1 contributes to the antioxidant defense mechanism by positively regulating the expression of the antioxidant genes under stress conditions in transgenic tobacco plants. PpNBS1 expressing transgenic tobacco plants resulted in lesser membrane damage, lower lipid peroxidation level, and higher chlorophyll content under stress conditions. Taken together, we conclude in addition to its known role as DNA damage sensor, PpNBS1 also plays a definite role in oxidative stress mitigation by minimizing ROS accumulation in the cell. • NBS1, a crucial member of plant DNA damage repair machinery contributes to ROS homeostasis in moss Physcomitrium. • Overexpression of PpNBS1 improves oxidative stress tolerance in transgenic plants by lowering cellular ROS level. • Constitutive expression of PpNBS1 induces antioxidant gene expression resulting in improved oxidative damage mitigation. [ABSTRACT FROM AUTHOR]

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required. [ABSTRACT FROM AUTHOR]

In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage. [ABSTRACT FROM AUTHOR]

p53 is primarily known as a downstream transcriptional effector in the DNA damage-response cascade. We report that endogenous p53 rapidly accumulates at DNA damage sites within 2 s of UVA microirradiation. The kinetics of p53 recruitment mimics those of known DNA damage-response proteins, such as Ku70 and poly(- ADP-ribose) polymerase (PARP), and precedes recruitment of Nbs1, 53BP1, and DDB1. Mutations in the DNA-binding and C-terminal domains significantly suppress this rapid recruitment. The C-terminal domain of p53 contains key residues for PARP interac- tion that are required for rapid recruitment of p53 to DNA damage sites, as is PARP-dependent modification. The presence of p53 at damage sites influences the recruitment kinetics of 53BP1 and DDB1 and directs the choice of nonhomologous end joining repair (NHEJ) and nucleotide excision repair. Mutations that suppressed rapid recruitment of p53 promoted error-prone alternative end- joining (alt-NHEJ) and inhibited nucleotide excision repair. Our finding that p53 is a critical early responder to DNA damage stands in contrast with its extensively studied role as a downstream transcriptional regulator in DNA damage repair. We highlight an unrecognized role of p53 in directing DNA repair dynamics and integrity and suggest a parallel mode of p53 tumor suppression apart from its function as a transcription factor. [ABSTRACT FROM AUTHOR]

We report a 62-year-old patient with uterine carcinosarcoma associated with a germline mutation in the NBN gene which is involved in the homologous recombination repair (HRR) pathway. This patient responded well to several different treatment strategies including platinum-based chemotherapy twice, PARP inhibitor therapy and immunotherapy, and is currently alive and with disease control, more than four years after diagnosis. This case is the first report of uterine carcinosarcoma associated with a germline mutation in NBN and highlights how specific genomic alterations may guide treatment decisions that may alter the natural history of an otherwise devastating disease.

DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and cancer. Central to the sensing of DSBs is the ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase- related protein kinase (PIKK) family. In response to DSBs, ATM is activated by the MRN (Mre11-Rad50- Nbs1) protein complex through a poorly understood process that also requires double-stranded DNA. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning of the N and C lobes along with relieving the blockage of the substrate-binding site. We also show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these structural findings with mutagenesis and biochemical assays. [ABSTRACT FROM AUTHOR]

MRE11, RAD50, and NBS1 form the MRN complex in response to DNA damage to activate ATM, a gene responsible for Ataxia-Telangiectasia (A-T). Loss of any components of the MRN complex compromises cell life. Mutations in MRE11 , RAD50 , and NBS1 cause human genomic instability syndromes Ataxia-Telangiectasia-like disorder (A-TLD), NBS-like disorder (NBSLD), and Nijmegen Breakage Syndrome (NBS), respectively. Among other pathologies, neuronal deficits, including microcephaly, intellectual disabilities, and progressive cerebellar degeneration, are common in these disorders. Nbs1 deletion in neural stem cells of mouse models resulted in cerebellar atrophy and ataxia, mimicking the A-T syndrome suggesting an etiological function of MRN-mediated DDR in neuronal homeostasis and neuropathology. Here we show that deletion of Nbs1 or Mre11 specifically in Purkinje neurons of mouse models (Nbs1 -PCΔ and Mre11 -PCΔ, respectively) is compatible with cerebellar development. Deleting Nbs1 in Purkinje cells disrupts the cellular localization pattern of MRE11 or RAD50 without inducing apparent DNA damage, albeit impaired DNA damage response (judged by 53BP1 focus formation) to ionizing radiation (IR). However, neither survival nor morphology of Purkinje cells and thus locomotor capabilities is affected by Nbs1 deletion under physiological conditions. Similarly, deletion of Mre11 in Purkinje cells does not affect the numbers or morphology of Purkinje cells and causes no accumulation of DNA damage. Mre11 -deleted Purkinje cells have regular intrinsic neuronal activity. Taken together, these data indicate that the MRN complex is not essential for the survival and functionality of postmitotic neurons such as Purkinje cells. Thus, cerebellar deficits in MRN defect-related disorders and mouse models are unlikely to be a direct consequence of loss of these factors compromising DDR in postmitotic neurons such as Purkinje cells. [ABSTRACT FROM AUTHOR]

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions. Author summary: ATRX is a protein that is needed to keep repetitive DNA regions organized. It does so in part by binding the DAXX histone chaperone to deposit histone proteins on DNA and assemble structures known as nucleosomes. While important, ATRX has additional functions that remain understudied. To better understand its various biological roles, we first identified the other proteins that are found in its proximity. ATRX-associating proteins were implicated in a range of functions, in addition to histone deposition. Our results suggest that ATRX-associating proteins likely help compact DNA after it is assembled into nucleosomes, and also promote its stability. We then examined the effect of ATRX on telomeres (repetitive DNA regions at the end of chromosomes). ATRX and at least one of its associating proteins suppressed spurious DNA exchanges at telomeres. To understand why, we then identified proteomic changes that occur at telomeres when ATRX was deleted. Loss of ATRX altered the enrichment of a surprising number of proteins at telomeres, including several DNA damage response and chromatin remodelling proteins. [ABSTRACT FROM AUTHOR]

In eukaryote cells, core components of chromatin, such as histones and DNA, are packaged in nucleus. Leakage of nuclear materials into cytosol will induce pathological effects. However, the underlying mechanisms remain elusive. Here, cytoplasmic localization of nuclear materials induced by chromatin dysregulation (CLIC) in mammalian cells is reported. H3K9me3 inhibition by small chemicals, HP1α knockdown, or knockout of H3K9 methylase SETDB1, induces formation of cytoplasmic puncta containing histones H3.1, H4 and cytosolic DNA, which in turn activates inflammatory genes and autophagic degradation. Autophagy deficiency rescues H3 degradation, and enhances the activation of inflammatory genes. MRE11, a subunit of MRN complex, enters cytoplasm after heterochromatin dysregulation. Deficiency of MRE11 or NBS1, but not RAD50, inhibits CLIC puncta in cytosol. MRE11 depletion represses tumor growth enhanced by HP1α deficiency, suggesting a connection between CLIC and tumorigenesis. This study reveals a novel pathway that heterochromatin dysregulation induces translocation of nuclear materials into cytoplasm, which is important for inflammatory diseases and cancer. [ABSTRACT FROM AUTHOR]

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex. [ABSTRACT FROM AUTHOR]

PurposeSevere combined immunodeficiency (SCID) encompasses a group of disorders characterized by reduced or absent T-cell number and function and identified by newborn screening utilizing T-cell receptor excision circles (TRECs). This screening has also identified infants with T lymphopenia who lack mutations in typical SCID genes. We report an infant with low TRECs and non-SCID T lymphopenia, who proved upon whole exome sequencing to have Nijmegen breakage syndrome (NBS).MethodsExome sequencing of DNA from the infant and his parents was performed. Genomic analysis revealed deleterious variants in the NBN gene. Confirmatory testing included Sanger sequencing and immunoblotting and radiosensitivity testing of patient lymphocytes.ResultsTwo novel nonsense mutations in NBN were identified in genomic DNA from the family. Immunoblotting showed absence of nibrin protein. A colony survival assay demonstrated radiosensitivity comparable to patients with ataxia telangiectasia.ConclusionsAlthough TREC screening was developed to identify newborns with SCID, it has also identified T lymphopenic disorders that may not otherwise be diagnosed until later in life. Timely identification of an infant with T lymphopenia allowed for prompt pursuit of underlying etiology, making possible a diagnosis of NBS, genetic counseling, and early intervention to minimize complications.

Abstract Background Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. We aimed to elucidate the impact of hypoxia on the sensitivity of MB cells to chemo- and radiotherapy. Methods We used two MB cell line (D283-MED and MEB-Med8A) and a widely used glioblastoma cell line (U87MG) for comparison. We applied a range of molecular and cellular techniques to measure cell survival, cell cycle progression, protein expression and DNA damage combined with a transcriptomic micro-array approach in D283-MED cells, for global gene expression analysis in acute and chronic hypoxic conditions. Results In D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to chemotherapy and X-ray irradiation. This acquired resistance upon chronic hypoxia was present but less pronounced in MEB-Med8A cells. Using transcriptomic analysis in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated (ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway. Conclusion Together our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB in D283-MED cells, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols.

The human genetic information has been orchestrated by DNA-damage response pathways. DNA damage response, DNA repair, programmed apoptosis and cell cycle progession are responsible for maintaining the genomic integrity, they initiates the process of tumorigenesis if they are not regulated properly. An article research was done through Google Scholar, Pubmed and was used for an electronic search of articles, using the following keywords: Double stranded breakage, Nibrin, P95, NBS1.The aim of the review is to point out the impact of double strand breakage and the role of Nibrin in development of cancers. The double strand breakage are pathological insults for all the microorganisms because they can cause the elimination of the essential genetic information, which results in the destruction of DNA replication Nibrin is the first DNA repair gene whose overexpression can promotes the process of tumorigenesis by the activation of phosphoinositol 3 kinase/akt pathways. [ABSTRACT FROM AUTHOR]

Introduction : The NBN gene product is part of the MRE11/RAD50/ NBN complex, which plays an essential role in genomic stability. In the study we try to answer the question what is the effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215W NBN gene mutations. Material and methods : Immortalized B-lymphocytes with NBN gene mutations were X-ray irradiated at doses of 1, 2, 5 and 8 Gy/min. Radioresistant DNA synthesis rate and the percentage of cells in phase S was analyzed by 3H thymidine and BrdU incorporation assays. NBN gene expression was quantified by real-time PCR with TaqMan fluorescent probe. Results : Increasing the irradiation dose resulted in gradual decrease of 3H thymidine incorporation in all cells, but significantly only in homo- and heterozygous c.657-661del cells (p-values < 0.0001). After irradiation the relative expression of NBN was significantly higher in homozygous c.657-661del and heterozygous p.R215W cells as compared to heterozygous c.657-661del, p.I171V and control cells (p < 0.01). All cells with NBN gene mutations showed significantly higher total number of chromosomal aberrations per metaphase as compared to control cells, with the highest number of aberrations in homozygous c.657-661del cells (p < 0.001). Conclusions : The results obtained indicate that homozygous c.657-661del mutation affects cell sensitivity to irradiation. Moreover, homozygous variant is associated with disturbance in the activation of cell cycle checkpoints and with defects in DNA repair. In turn, heterozygous c.657-661del, p.R215W and p.I171V mutations do not substantially alter the radiosensitivity.

The NBN gene has been included in breast cancer (BC) multigene panels based on early studies suggesting an increased BC risk for carriers, though not confirmed by recent research. To evaluate the impact of NBN analysis, we assessed the results of NBN sequencing in 116 BRCA-negative BC patients and reviewed the literature. Three patients (2.6%) carried potentially relevant variants: two, apparently unrelated, carried the frameshift variant c.156_157delTT and another one the c.628G>T variant. The latter was subsequently found in 4/1390 (0.3%) BC cases and 8/1580 (0.5%) controls in an independent sample, which, together with in silico predictions, provided evidence against its pathogenicity. Conversely, the rare c.156_157delTT variant was absent in the case-control set; moreover, a 50% reduction of NBN expression was demonstrated in one carrier. However, in one family it failed to co-segregate with BC, while the other carrier was found to harbor also a probably pathogenic TP53 variant that may explain her phenotype. Therefore, the c.156_157delTT, although functionally deleterious, was not supported as a cancer-predisposing defect. Pathogenic/likely pathogenic NBN variants were detected by multigene panels in 31/12314 (0.25%) patients included in 15 studies. The risk of misinterpretation of such findings is substantial and supports the exclusion of NBN from multigene panels.

Background: Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder leading to chromosomal instability and an array of symptoms, including characteristic facial features (bird-like face), predisposition to malignancies, as well as hypergonadotropic hypogonadism. This case report discusses the diagnostic process and management of a 23-year-old Polish female patient who was admitted to hospital with symptoms of secondary amenorrhea and clinical features corresponding to NBS. Methods: Clinical examination, per-rectal ultrasound, laboratory diagnostics (including serum concentrations of FSH, LH, estradiol, testosterone, and TSH), as well as SSCP analysis and classic karyotyping were performed. Results: During hormonal evaluation elevated serum concentration of FSH and LH and decreased serum concentration of estradiol were measured. The genetic testing revealed translocation 7;14 (t(7;14)) and inversion 7 in 22% of examined cells which confirmed the initial hypothesis of NBS. The diagnosis was finally verified by identifying a Slavic founder mutation, c.657_661del5, on both allels of the NBN gene. Furthermore, hormonal serum evaluation conducted after four weeks allowed the patient to be diagnosed with premature ovarian insufficiency (POI) suspected earlier on the grounds of preliminary examinations (ultrasound imaging and laboratory tests). Conclusions: Chromosomal instability resulting from a mutation present in Nijmegen breakage syndrome patients might be a causative factor of premature ovarian insufficiency. Therefore, females diagnosed with NBS should undergo additional diagnostic procedures in order to determine further management and treatment. [ABSTRACT FROM AUTHOR]

Background: Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. We aimed to elucidate the impact of hypoxia on the sensitivity of MB cells to chemo- and radiotherapy.Methods: We used two MB cell line (D283-MED and MEB-Med8A) and a widely used glioblastoma cell line (U87MG) for comparison. We applied a range of molecular and cellular techniques to measure cell survival, cell cycle progression, protein expression and DNA damage combined with a transcriptomic micro-array approach in D283-MED cells, for global gene expression analysis in acute and chronic hypoxic conditions.Results: In D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to chemotherapy and X-ray irradiation. This acquired resistance upon chronic hypoxia was present but less pronounced in MEB-Med8A cells. Using transcriptomic analysis in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated (ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway.Conclusion: Together our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB in D283-MED cells, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols. [ABSTRACT FROM AUTHOR]

Platinum (Pt) compounds are an important class of anti-cancer therapeutics, but outstanding questions remain regarding their mechanism of action. Here, we demonstrate that oxaliplatin, a Pt drug used to treat colorectal cancer, inhibits rRNA transcription through ATM and ATR signaling, and induces DNA damage and nucleolar disruption. We show that oxaliplatin causes nucleolar accumulation of the nucleolar DNA damage response proteins (n-DDR) NBS1 and TOPBP1; however transcriptional inhibition does not depend upon NBS1 or TOPBP1, nor does oxaliplatin induce substantial amounts of nucleolar DNA damage, distinguishing the nucleolar response from previously characterized n-DDR pathways. Taken together, our work indicates that oxaliplatin induces a distinct ATM and ATR signaling pathway that functions to inhibit Pol I transcription in the absence of direct nucleolar DNA damage, demonstrating how nucleolar stress and transcriptional silencing can be linked to DNA damage signaling and highlighting an important mechanism of Pt drug cytotoxicity. [Display omitted] • Oxaliplatin specifically inhibits RNA Pol I and disrupts nucleolar morphology • Oxaliplatin induces n-DDR factor recruitment in the absence of nucleolar DNA damage • Inhibition of nucleolar transcription by oxaliplatin involves ATM and ATR signaling • NBS1 is not required for nucleolar RNA Pol I inhibition by oxaliplatin Oxaliplatin is an important chemotherapeutic known to induce nucleolar stress and inhibit RNA polymerase I, distinct from the related Pt drug cisplatin. Nechay et al. demonstrate that inhibition of nucleolar transcription by oxaliplatin involves DNA damage signaling through ATM/ATR kinases in the absence of nucleolar DNA damage. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes (MDS) are clonal hematologic stem cell disorders with an as yet unknown molecular pathology. Genetic instability has been proposed as a cause of MDS. Mutations in the NBS1 gene, whose product nibrin (p95) is involved in DNA damage repair and cell-cycle control, might be associated with an elevated predisposition to the development of MDS. The aim of the study was to examine truncating 5 bp deletion (657del5), the most frequent NBS1 gene mutation in Slavic populations, in MDS patients. Among 71 MDS patients, we found one case that was heterozygous for the NBS1 657del5 mutation. To the best of our knowledge, this is the first report of a NBS1 mutation in MDS. [Projekat Ministarstva nauke Republike Srbije, br. 175091]

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms. [ABSTRACT FROM AUTHOR]

Introduction: The NBN gene product is part of the MRE11/RAD50/NBN complex, which plays an essential role in genomic stability. In the study we try to answer the question what is the effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215W NBN gene mutations.Material and Methods: Immortalized B-lymphocytes with NBN gene mutations were X-ray irradiated at doses of 1, 2, 5 and 8 Gy/min. Radioresistant DNA synthesis rate and the percentage of cells in phase S was analyzed by 3H thymidine and BrdU incorporation assays. NBN gene expression was quantified by real-time PCR with TaqMan fluorescent probe.Results: Increasing the irradiation dose resulted in gradual decrease of 3H thymidine incorporation in all cells, but significantly only in homo- and heterozygous c.657-661del cells (p-values < 0.0001). After irradiation the relative expression of NBN was significantly higher in homozygous c.657-661del and heterozygous p.R215W cells as compared to heterozygous c.657-661del, p.I171V and control cells (p < 0.01). All cells with NBN gene mutations showed significantly higher total number of chromosomal aberrations per metaphase as compared to control cells, with the highest number of aberrations in homozygous c.657-661del cells (p < 0.001).Conclusions: The results obtained indicate that homozygous c.657-661del mutation affects cell sensitivity to irradiation. Moreover, homozygous variant is associated with disturbance in the activation of cell cycle checkpoints and with defects in DNA repair. In turn, heterozygous c.657-661del, p.R215W and p.I171V mutations do not substantially alter the radiosensitivity. [ABSTRACT FROM AUTHOR]

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. In this work we examine the interaction between TOPBP1 and NBS1 in detail. We report that NBS1 uses its BRCT1 domain to interact with TOPBP1′s BRCT1 domain and, separately, with TOPBP1′s BRCT2 domain. Thus, NBS1 can make two distinct contacts with TOPBP1. We report that recombinant TOPBP1 and NBS1 proteins bind one another in a purified system, showing that the interaction is direct and does not require post-translational modifications. Surprisingly, we also report that intact BRCT domains are not required for these interactions, as truncated versions of the domains are sufficient to confer binding. For TOPBP1, we find that small 24–29 amino acid sequences within BRCT1 or BRCT2 allow binding to NBS1, in a transferrable manner. These data expand our knowledge of how the crucial DNA damage response proteins TOPBP1 and NBS1 interact with one another and set the stage for functional analysis of the two disparate binding sites for NBS1 on TOPBP1. • TOPBP1 and NBS1 interact with one another. • The TOPBP1::NBS1 interaction is driven by their BRCT domains. • The NBS1 BRCT1 domain can bind to TOPBP1′s BRCT1 domain and to TOPBP1′s BRCT2 domain. • The TOPBP1::NBS1 interaction is direct and does not require post-translational modifications. • Small peptides within TOPBP1′s BRCT1 and BRCT2 domains can confer binding to NBS1 onto a heterologous protein. [ABSTRACT FROM AUTHOR]

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions. [Display omitted] • Cryo-EM structure of full-length, eukaryotic Mre11-Rad50-Nbs1 bound to ATPγS • A single Nbs1 polypeptide wraps around and stabilizes the Mre11 dimer • ATP-dependent and -independent binding modes for DNA ends and internal DNA • Rad50 coiled-coil apex tetramerization forms MRN-MRN tethering structures Rotheneder et al. determine the structure of eukaryotic Mre11-Rad50-Nbs1, reveal the basis for Nbs1 interaction with the Mre11 dimer, uncover ATP-dependent and -independent DNA-binding modes for DNA ends and internal DNA, and reveal that Rad50 coiled-coil apex tetramers form DNA-tethering structures in DNA double-strand break repair. [ABSTRACT FROM AUTHOR]

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (−/−) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (−/−) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (−/−) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR. [ABSTRACT FROM AUTHOR]