NBS1 Protein Research Articles

NBS1 dePARylation by NUDT16 is critical for DNA double-strand break repair.

NBS1, a protein linked to the autosomal recessive disorder Nijmegen breakage syndrome, plays an essential role in the DNA damage response and DNA repair. Despite its importance, the mechanisms regulating NBS1 and the impact of this regulation on DNA repair processes remain obscure. In this study, we discovered a new post-translational modification of NBS1, ADP-ribosylation. This modification can be removed by the NUDT16 hydrolase. The loss of NUDT16 results in a reduction of NBS1 protein levels due to NBS1 PARylation-dependent ubiquitination and degradation, which is mediated by the PAR-binding E3 ubiquitin ligase, RNF146. Importantly, ADP-ribosylation of NBS1 is crucial for its localization at DSBs and its involvement in homologous recombination (HR) repair. Additionally, the NUDT16-NBS1 interaction is regulated in response to DNA damage, providing further rationale for NBS1 regulation by NUDT16 hydrolase. In summary, our study unveils the critical role of NUDT16 in governing both the stability of NBS1 and recruitment of NBS1 to DNA double-strand breaks, providing novel insights into the regulation of NBS1 in the HR repair pathway.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Inhibition of nucleolar transcription by oxaliplatin involves ATM/ATR kinase signaling

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.

NBS1 binds directly to TOPBP1 via disparate interactions between the NBS1 BRCT1 domain and the TOPBP1 BRCT1 and BRCT2 domains

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.

A gene dosage-dependent effect unveils NBS1 as both a haploinsufficient tumour suppressor and an essential gene for SHH-medulloblastoma.

AimsInherited 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 methodsWe 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.ResultsWe 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 Nbn KO impairs MB development both in SmoA1 mice and in an SHH‐driven tumour allograft.ConclusionsOur study indicates that Nbn is haploinsufficient for SHH‐MB development whereas full Nbn KO is epistatic on SHH‐driven MB development, thus revealing a gene dosage‐dependent effect of Nbn inactivation on SHH‐MB development.

In silico comprehensive analysis of coding and non-coding SNPs in human mTOR protein.

The mammalian/mechanistic target of rapamycin (mTOR) protein is an important growth regulator and has been linked with multiple diseases including cancer and diabetes. Non-synonymous mutations of this gene have already been found in patients with renal clear cell carcinoma, melanoma, and acute lymphoid leukemia among many others. Such mutations can potentially affect a protein’s structure and hence its functions. In this study, therefore, the most deleterious SNPs of mTOR protein have been determined to identify potential biomarkers for various disease treatments. The aim is to generate a structured dataset of the mTOR gene’s SNPs that may prove to be an asset for the identification and treatment of multiple diseases associated with the target gene. Both sequence and structure-based approaches were adopted and a wide variety of bioinformatics tools were applied to analyze the SNPs of mTOR protein. In total 11 nsSNPs have been filtered out of 2178 nsSNPs along with two non-coding variations. All of the nsSNPs were found to destabilize the protein structure and disrupt its function. While R619C, A1513D, and T1977R mutations were shown to alter C alpha distances and bond angles of the mTOR protein, L509Q, R619C and N2043S were predicted to disrupt the mTOR protein’s interaction with NBS1 protein and FKBP1A/rapamycin complex. In addition, one of the non-coding SNPs was shown to alter miRNA binding sites. Characterizing nsSNPs and non-coding SNPs and their harmful effects on a protein’s structure and functions will enable researchers to understand the critical impact of mutations on the molecular mechanisms of various diseases. This will ultimately lead to the identification of potential targets for disease diagnosis and therapeutic interventions.

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3′−5′ exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.

Adjacent mutations in the archaeal Rad50 ABC ATPase D-loop disrupt allosteric regulation of ATP hydrolysis through different mechanisms.

DNA damage is the driving force for mutation and genomic instability, which can both lead to cell death or carcinogenesis. DNA double strand breaks are detected and processed in part by the Mre11–Rad50–Nbs1 protein complex. Although the Mre11–Rad50–Nbs1 complex is essential, several spontaneous mutations have been noted in various cancers. One of these mutations, within a conserved motif of Rad50, resulted in an outlier curative response in a clinical trial. We show through biochemical and biophysical characterization that this cancer-associated mutation and a second mutation to the adjacent residue, previously described in a breast cancer patient, both have gain-of-function Rad50 ATP hydrolysis activity that results not from faster association of the ATP-bound form but faster dissociation leading to less stable Rad50 dimer. This disruption impairs the regulatory functions of the protein complex leading to a loss of exonuclease activity from Mre11. Interestingly, these two mutations affect Rad50 structure and dynamics quite differently. These studies describe the relationship between function, structure, and molecular motions in improperly regulated Rad50, which reveal the underlying biophysical mechanism for how these two cancer-associated mutations affect the cell.

Abstract A103: Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance

Abstract Resistance to radiation and chemotherapy in colorectal patients is considered one of the major contributors of refractory disease and disease progression. Dysregulation or aberrant activation of the SHH/GLI1 signaling pathway, common to both colorectal cancer and other cancer subtypes, influences the integrity of the DNA damage response (DDR) pathway, which may impact tumor response to chemo- and radiation therapies. The DDR pathway is initiated in part by the recognition of a DNA lesion by a sensor protein and the recruitment NBS1 to that lesion. Subsequent repair is initiated by the Mre11, Rad50, NBS1 (MRN) complex formation. In this study, we investigated the expression of GLI1 and NBS1 in tissues samples from 188 patients diagnosed with colorectal cancer by immunohistochemistry and analyzed the clinical significance and prognostic relevance of elevated GLI1-NBS1 co-expression. GLI1 expression was positively associated with the NBS1 expression, and high expression of both in the biopsied specimens significantly correlated with poor patient survival. The result of a series of biochemical experiments directly links GLI1 transcriptional activity to the DNA damage repair pathway. First, NBS1 mRNA levels and protein were found to be reduced upon treatment with GANT61, a GLI inhibitor, indicating that GLI activity was required for NBS1 transcription. Second, overexpression of NBS1 in HT29 cells rescued GANT61-induced cell death by measure of cleaved caspase-3, indicating a significant role of induced DNA damage in the mechanism of GANT61-induced cell death in oncogenic GLI1 cancers. Third, this was verified by ChIP analysis of GLI1 binding to the NBS1 promotor region containing a putative GLI binding sequence. The efficacy of GLI1 inhibition as therapeutic strategy was then tested in parallel with 5-Fluorouracil (5-FU), a component of the standard-of-care therapy FOLFOX. In vitro, colorectal cell lines with GLI1-driven NBS1 expression demonstrated strong 5-FU resistance. These cells were re-sensitized to 5-FU by silencing the expression of NBS1 with siRNA, or with combination therapy involving 5-FU and GANT61. This suggests that a first-in class chemotherapeutic strategy may provide additional treatment options for patients demonstrating 5-FU resistance during the course of their chemotherapy. While GANT61 is commonly used to inhibit GLI in vitro, it exhibits poor pharmacokinetic properties and is not a viable drug for the clinic. To that end, we present a novel GLI1 antagonist SR38832, which exhibits a more stable pharmacokinetic profile than GANT61 in xenograft mouse models and lower IC50 in colon cancer cell lines. In a human colorectal cancer xenograft mouse model, SR38832 significantly inhibited both tumor growth and proliferation. Not only has the identification of SR38832 provided an in vivo tool to study GLI oncogenic activity, but SR38832 is also structural lead to be optimized through an iterative medicinal chemistry approach. Citation Format: Ruowen Zhang, Justin Avery, Jinlu Ma, Theresa Nguyen, Mark J Suto, Bo Xu, Rebecca J Boohaker. Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A103. doi:10.1158/1535-7163.TARG-19-A103

Overexpression of the MRE11-RAD50-NBS1 (MRN) complex in rectal cancer correlates with poor response to neoadjuvant radiotherapy and prognosis

BackgroundThe MRE11/RAD50/NBS1 (MRN) complex plays an essential role in detecting and repairing double-stranded breaks, and thus the potential roles of MRE11, RAD50 and NBS1 proteins in the pathogenesis of various cancers is the subject of investigation. This study was aimed at assessing the three-protein panel of MRN complex subunits as a potential radiosensitivity marker and evaluating the prognostic and clinicopathological implications of MRN expression in rectal cancer.MethodsSamples from 265 rectal cancer patients treated with surgery and adjuvant chemoradiotherapy, including samples from 55 patients who were treated with neoadjuvant radiotherapy between 2000 and 2011, were analyzed. Expression of MRN complex proteins in tissue samples was determined by immunohistochemistry. Univariate and multivariate analyses were carried out to identify clinicopathological characteristics that are associated with the MRN three-protein panel expression in rectal cancer samples.ResultsIn Kaplan–Meier survival analyses, we found that high level expression of MRN complex proteins in postoperative samples was associated with poor disease-free (p = 0.021) and overall (P = 0.002) survival. Interestingly, high MRN expression also correlated with poor disease-free (P = 0.047) and overall (P = 0.024) survival in the neoadjuvant radiotherapy subgroup. In multivariate analysis, combined MRN expression (hazard ratio = 2.114, 95% confidence interval 1.096–4.078, P = 0.026) and perineural invasion (hazard ratio = 2.160, 95% confidence interval 1.209–3.859, P = 0.009) were significantly associated with a worse disease-free survival.ConclusionsExpression levels of MRN complex proteins significantly predict disease-free survival in rectal cancer patients, including those treated with neoadjuvant radiotherapy, and may have value in the management of these patients.

LSD1 mediated changes in the local redox environment during the DNA damage response.

The redox state of the cell can be affected by many cellular conditions. In this study we show that detectable reactive oxygen species (ROS) are also generated in response to DNA damage by the chromatin remodeling factor and monoamine oxidase LSD1/KDM1A. This raised the possibility that the localized generation of hydrogen peroxide produced by LSD1 may affect the function of proximally located DNA repair proteins. The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and non-homologous end joining (NHEJ). Cells were exposed to low levels of ectopic H2O2, DNA breaks generated by laser light, and recruitment kinetics of NHEJ protein Ku80 to DNA damage sites determined. Ku80 recruitment to damage sites was significantly decreased in cells pretreated with H2O2 while HR end binding protein Nbs1 was increased. This suggests that the DNA repair pathway choice has the potential to be modulated by the local redox state. This has implications for chemotherapeutic approaches involving generating DNA damage to target actively dividing cancer cells, which may be more or less effective dependent on the redox state of the targeted cells and the predominant repair pathway required to repair the type of DNA damage generated.

The Major Tegument Protein of Bovine Herpesvirus 1, VP8, Interacts with DNA Damage Response Proteins and Induces Apoptosis

VP8, the UL47 gene product in bovine herpesvirus-1 (BoHV-1), is a major tegument protein that is essential for virus replication in vivo The major DNA damage response protein, ataxia telangiectasia mutated (ATM), phosphorylates Nijmegen breakage syndrome (NBS1) and structural maintenance of chromosome-1 (SMC1) proteins during the DNA damage response. VP8 was found to interact with ATM and NBS1 during transfection and BoHV-1 infection. However, VP8 did not interfere with phosphorylation of ATM in transfected or BoHV-1-infected cells. In contrast, VP8 inhibited phosphorylation of both NBS1 and SMC1 in transfected cells, as well as in BoHV-1-infected cells, but not in cells infected with a VP8 deletion mutant (BoHV-1ΔUL47). Inhibition of NBS1 and SMC1 phosphorylation was observed at 4 h postinfection by nuclear VP8. Furthermore, UV light-induced cyclobutane pyrimidine dimer (CPD) repair was reduced in the presence of VP8, and VP8 in fact enhanced etoposide or UV-induced apoptosis. This suggests that VP8 blocks the ATM/NBS1/SMC1 pathway and inhibits DNA repair. VP8 induced apoptosis in VP8-transfected cells through caspase-3 activation. The fact that BoHV-1 is known to induce apoptosis through caspase-3 activation is in agreement with this observation. The role of VP8 was confirmed by the observation that BoHV-1 induced significantly more apoptosis than BoHV-1ΔUL47. These data reveal a potential role of VP8 in the modulation of the DNA damage response pathway and induction of apoptosis during BoHV-1 infection.IMPORTANCE To our knowledge, the effect of BoHV-1 infection on the DNA damage response has not been characterized. Since BoHV-1ΔUL47 was previously shown to be avirulent in vivo, VP8 is critical for the progression of viral infection. We demonstrated that VP8 interacts with DNA damage response proteins and disrupts the ATM-NBS1-SMC1 pathway by inhibiting phosphorylation of DNA repair proteins NBS1 and SMC1. Furthermore, interference of VP8 with DNA repair was correlated with decreased cell viability and increased DNA damage-induced apoptosis. These data show that BoHV-1 VP8 developed a novel strategy to interrupt the ATM signaling pathway and to promote apoptosis. These results further enhance our understanding of the functions of VP8 during BoHV-1 infection and provide an additional explanation for the reduced virulence of BoHV-1ΔUL47.

Regulation of c-Myc and NBS1 by ionizing radiation in glioblastoma cells

Glioblastoma is the most common primary brain tumor presenting the worst prognostic. Radioresistance of these tumors is barrier to better treatment and management. Cellular response to ionizing radiation (IR) depends on various molecular mechanisms, including transcription of DNA damage response genes in response to oncogenes. Previous studies suggested that the radioresistance of glioma stem cells would be associated with c-Myc-dependent upregulation of NBS1. The purpose of this study was to evaluate whether IR and other DNA-damaging agents co-regulate c-Myc and NBS1 in glioblastoma cell lines. We found that c-Myc protein level is downregulated by IR in specific glioblastoma cell lines, while NBS1 protein content was not altered in response to genotoxic agents. Additionally, IR-induced downregulation of c-Myc protein content had no correlation with decreased cell viability. These findings indicate that the functional relationship between c-Myc and NBS1 in the context of genotoxic stress may be genetic background-specific.

DNA damage and repair proteins in cellular response to sulfur mustard in Iranian veterans more than two decades after exposure

Delayed effects of sulfur mustard (SM) exposure on the levels of five important damage/repair proteins were investigated in 40 SM-exposed veterans of Iran-Iraq war and 35 unexposed controls. A major DNA damage biomarker protein – phosphorylated H2AX – along with four DNA repair proteins in cell response to the genome damage MRE11, NBS1, RAD51, and XPA were evaluated in blood lymphocytes from the veterans and controls using western blotting. Mean levels of XPA, MRE11, RAD51 and NBS1 were lower in SM-exposed patients and the decrease in NBS1 was significant. Even though the raised level of phosphor-H2AX in SM-poisoned group compared to the controls was not significant it was consistent with DNA damage findings confirming the severity of damage to the DNA after exposure to SM. There were correlations between the values of RAD51 and NBS1 proteins as well as XPA and MRE11 proteins. More than two decades after exposure to SM, there is still evidences of DNA damage as well as impaired repair mechanisms in cells of exposed individuals. Such disorders in cellular level may contribute to long term health problems of the SM veterans.

Lack of MRE11-RAD50-NBS1 (MRN) complex detection occurs frequently in low-grade epithelial ovarian cancer

BackgroundBRCA1/2-deficient ovarian carcinomas are recognized as target for Poly (ADP-ribose) polymerase (PARP) inhibitors. BRCA1 and BRCA2 proteins are involved in homologous recombination repair of double-strand DNA breaks. The relevance of other homologous recombination repair proteins, e.g. MRE11, RAD50, NBS1 (MRN complex) in ovarian carcinomas is unclear. The objective of this study was to investigate the prevalence of lack of MRE11, RAD50, NBS1 protein detection in epithelial ovarian cancer (EOC).MethodsA tissue microarray (TMA) with 134 EOC was immunohistochemically evaluated for MRE11, RAD50 and NBS1. Data was analysed for associations with clinicopathological parameters, histological subtype, patient overall survival and mismatch repair (MMR) protein status. Sensitivity towards the PARP inhibitor BMN673 was tested in two ovarian cancer cell lines (TOV-21 and OVTOKO) using colony formation assays.ResultsLack of MRN complex protein detection was seen in 41% (55/134) of EOC and was more frequent in low-grade (57.6%; 19/33) than in high-grade EOC (18.8%; 36/101; n = 134; p = 0.04). There was an association with the ovarian carcinoma subtype (60.3%; 35/58 lack of detection in type I versus 26.3%; 20/76 in type II; n = 134; p < 0.001) as well as undetectable DNA mismatch repair proteins MLH1 and MSH2 (89.3%; 25/28; n = 131; p < 0.001). MRE11 knockdown led to moderately increased sensitivity towards the PARP inhibitor BMN673 in one ovarian carcinoma cell line in vitro.ConclusionsFrequent lack of MRE11, RAD50, NBS1 protein detection in type I human ovarian carcinomas is observed in EOC and our data suggests further investigation regarding sensitivity to PARP-inhibition in tumours lacking MRE11 expression.

The Mre11-Nbs1 Interface Is Essential for Viability and Tumor Suppression

The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. Todelineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108-amino-acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and support differentiation of hematopoietic cells invivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11 and that most of the Nbs1 protein is dispensable for Mre11 complex functions and suggest that Mre11 and Rad50 directly activate ATM.

A balance between elongation and trimming regulates telomere stability in stem cells.

Telomere length maintenance ensures self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), however the mechanisms governing telomere length homeostasis in these cell types are unclear. Here, we report that telomere length is determined by the balance between telomere elongation mediated by telomerase and telomere trimming, controlled by the homologous recombination proteins XRCC3 and Nbs1 that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles), respectively. We found that reprogramming of differentiated cells induces T-circle and single stranded C-rich telomeric DNA accumulation, indicating the activation of telomere trimming pathways that compensate telomerase dependent telomere elongation in hiPSCs. Excessive telomere elongation compromises telomere stability and promotes the formation of partially single-stranded telomeric DNA circles (C-circles) in hESCs, suggesting heightened sensitivity of stem cells to replication stress at overly long telomeres. Thus, tight control of telomere length homeostasis is essential to maintain telomere stability in hESCs.

Nucleolar Reorganization Upon Site-Specific Double-Strand Break Induction.

DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.

An Intrinsically Disordered Region of the DNA Repair Protein Nbs1 Is a Species-Specific Barrier to Herpes Simplex Virus 1 in Primates.

Humans occasionally transmit herpes simplex virus 1 (HSV-1) to captive primates, who reciprocally harbor alphaherpesviruses poised for zoonotic transmission to humans. To understand the basis for the species-specific restriction of HSV-1 in primates, we simulated what might happen during the cross-species transmission of HSV-1 and found that the DNArepair protein Nbs1 from only some primate species is able to promote HSV-1 infection. The Nbs1 homologs that promote HSV-1 infection also interact with the HSV-1 ICP0 protein. ICP0 interaction mapped to a region of structural disorder in the Nbs1protein. Chimeras reversing patterns of disorder in Nbs1 reversed titers of HSV-1 produced in the cell. By extending this analysis to 1,237 virus-interacting mammalian proteins, we show that proteins that interact with viruses are highly enriched in disorder, suggesting that viruses commonly interact with host proteins through intrinsically disordered domains.

Chromosomal Instability and Molecular Defects in Induced Pluripotent Stem Cells from Nijmegen Breakage Syndrome Patients

Nijmegen breakage syndrome (NBS) results from theabsence of the NBS1 protein, responsible for detection of DNA double-strand breaks (DSBs). NBS is characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Here, we show successful reprogramming ofNBS fibroblasts into induced pluripotent stem cells (NBS-iPSCs). Our data suggest a strong selection for karyotypically normal fibroblasts to go throughthe reprogramming process. NBS-iPSCs then acquire numerous chromosomal aberrations and show a delayed response to DSB induction. Furthermore, NBS-iPSCs display slower growth, mitotic inhibition, a reduced apoptotic response tostress, and abnormal cell-cycle-related gene expression. Importantly, NBS neural progenitor cells (NBS-NPCs) show downregulation of neural developmental genes, which seems to be mediated by P53. Our results demonstrate the importance of NBS1 in early human development, shed light on the molecular mechanisms underlying this severe syndrome, and further expand our knowledge of the genomic stress cells experience during the reprogramming process.