ATR-mediated DNA Damage Response Research Articles

APE1 is a master regulator of the ATR-/ATM-mediated DNA damage response

To maintain genomic integrity, cells have evolved several conserved DNA damage response (DDR) pathways in response to DNA damage and stress conditions. Apurinic/apyrimidinic endonuclease 1 (APE1) exhibits AP endonuclease, 3′-5′ exonuclease, 3′-phosphodiesterase, and 3′-exoribonuclease activities and plays critical roles in the DNA repair and redox regulation of transcription. However, it remains unclear whether and how APE1 is involved in DDR pathways. In this perspective, we first updated our knowledge of APE1's functional domains and its nuclease activities and their specific associated substrates. We then summarized the newly discovered roles and mechanisms of action of APE1 in the global and nucleolar ATR-mediated DDR pathway. While the ATM-mediated DDR is well known to be activated by DNA double-strand breaks and oxidative stress, here we provided new perspectives as to how ATM DDR signaling is activated by indirect single-strand breaks (SSBs) resulting from genotoxic stress and defined SSB structures, and discuss how ATM kinase is directly activated and regulated by its activator, APE1. Together, accumulating body of new evidence supports the notion that APE1 is a master regulator protein of the ATR- and ATM-mediated DDR pathways. These new findings of APE1 in DDR signaling provide previously uncharacterized but critical functions and regulations of APE1 in genome integrity.

Mechanism of Musashi2 affecting radiosensitivity of lung cancer by modulating DNA damage repair.

Identifying new targets for overcoming radioresistance is crucial for improving the efficacy of lung cancer radiotherapy, given that tumor cell resistance is a leading cause of treatment failure. Recent research has spotlighted the significance of Musashi2 (MSI2) in cancer biology. In this study, we first demonstrated that MSI2 plays a key function in regulating the radiosensitivity of lung cancer. The expression of MSI2 is negatively correlated with overall survival in cancer patients, and the knockdown of MSI2 inhibits tumorigenesis and increases radiosensitivity of lung cancer cells. Cellular radiosensitivity, which is closely linked to DNA damage, is influenced by MSI2 interaction with ataxia telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1) post-irradiation; moreover, knockdown of MSI2 inhibits the ATR-mediated DNA damage response pathway. RNA-binding motif protein 17 (RBM17), which is implicated in DNA damage repair, exhibits increased interaction with MSI2 post-irradiation. We found that knockdown of RBM17 disrupted the interaction between MSI2 and ATR post-irradiation and increased the radiosensitivity of lung cancer cells. Furthermore, we revealed the potential mechanism of MSI2 recruitment into the nucleus with the assistance of RBM17 to activate ATR to promote radioresistance. This study provides novel insights into the potential application of MSI2 as a new target in lung cancer radiotherapy.

Nuclear ATR lysine-tyrosylation protects against heart failure by activating DNA damage response

Dysregulated amino acid increases the risk for heart failure (HF) via unclear mechanisms. Here, we find that increased plasma tyrosine and phenylalanine levels are associated with HF. Increasing tyrosine or phenylalanine by high-tyrosine or high-phenylalanine chow feeding exacerbates HF phenotypes in transverse aortic constriction and isoproterenol infusion mice models. Knocking down phenylalanine dehydrogenase abolishes the effect of phenylalanine, indicating that phenylalanine functions by converting to tyrosine. Mechanistically, tyrosyl-tRNA synthetase (YARS) binds to ataxia telangiectasia and Rad3-related gene (ATR), catalyzes lysine tyrosylation (K-Tyr) of ATR, and activates the DNA damage response (DDR) in the nucleus. Increased tyrosine inhibits the nuclear localization of YARS, inhibits the ATR-mediated DDR, accumulates DNA damage, and elevates cardiomyocyte apoptosis. Enhancing ATR K-Tyr by overexpressing YARS, restricting tyrosine, or supplementing tyrosinol, a structural analog of tyrosine, promotes YARS nuclear localization and alleviates HF in mice. Our findings implicate facilitating YARS nuclear translocation as a potential preventive and/or interfering measure against HF.

P53 triggers mitochondrial apoptosis following DNA damage-dependent replication stress by the hepatotoxin methyleugenol

Liver cancer is one of the most frequent tumor entities worldwide, which is causally linked to viral infection, fatty liver disease, life-style factors and food-borne carcinogens, particularly aflatoxins. Moreover, genotoxic plant toxins including phenylpropenes are suspected human liver carcinogens. The phenylpropene methyleugenol (ME) is a constituent of essential oils in many plants and occurs in herbal medicines, food, and cosmetics. Following its uptake, ME undergoes Cytochrome P450 (CYP) and sulfotransferase 1A1 (SULT1A1)-dependent metabolic activation, giving rise to DNA damage. However, little is known about the cellular response to the induced DNA adducts. Here, we made use of different SULT1A1-proficient cell models including primary hepatocytes that were treated with 1′-hydroxymethyleugenol (OH-ME) as main phase I metabolite. Firstly, mass spectrometry showed a concentration-dependent formation of N2-MIE-dG as major DNA adduct, strongly correlating with SULT1A1 expression as attested in cells with and without human SULT1A1. ME-derived DNA damage activated mainly the ATR-mediated DNA damage response as shown by phosphorylation of CHK1 and histone 2AX, followed by p53 accumulation and CHK2 phosphorylation. Consistent with these findings, the DNA adducts decreased replication speed and caused replication fork stalling. OH-ME treatment reduced viability particularly in cell lines with wild-type p53 and triggered apoptotic cell death, which was rescued by pan-caspase-inhibition. Further experiments demonstrated mitochondrial apoptosis as major cell death pathway. ME-derived DNA damage caused upregulation of the p53-responsive genes NOXA and PUMA, Bax activation, and cytochrome c release followed by caspase-9 and caspase-3 cleavage. We finally demonstrated the crucial role of p53 for OH-ME triggered cell death as evidenced by reduced pro-apoptotic gene expression, strongly attenuated Bax activation and cell death inhibition upon genetic knockdown or pharmacological inhibition of p53. Taken together, our study demonstrates for the first time that ME-derived DNA damage causes replication stress and triggers mitochondrial apoptosis via the p53-Bax pathway.

ATR-mediated DNA damage responses underlie aberrant B cell activity in systemic lupus erythematosus.

B cells orchestrate autoimmune responses in patients with systemic lupus erythematosus (SLE), but broad-based B cell-directed therapies show only modest efficacy while blunting humoral immune responses to vaccines and inducing immunosuppression. Development of more effective therapies targeting pathogenic clones is a currently unmet need. Here, we demonstrate enhanced activation of the ATR/Chk1 pathway of the DNA damage response (DDR) in B cells of patients with active SLE disease. Treatment of B cells with type I IFN, a key driver of immunity in SLE, induced expression of ATR via binding of interferon regulatory factor 1 to its gene promoter. Pharmacologic targeting of ATR in B cells, via a specific inhibitor (VE-822), attenuated their immunogenic profile, including proinflammatory cytokine secretion, plasmablast formation, and antibody production. Together, these findings identify the ATR-mediated DDR axis as the orchestrator of the type I IFN-mediated B cell responses in SLE and as a potential novel therapeutic target.

A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA

Background and AimsThe persistence of viral covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment against hepatitis B virus (HBV). Basic and translational studies are largely hampered due to the lack of feasible small animal models to support HBV cccDNA formation. The aim of this study is to establish a novel mouse model harboring cccDNA.MethodsAn adeno-associated virus (AAV) vector carrying a replication-deficient HBV1.04-fold genome (AAV-HBV1.04) was constructed. The linear HBV genome starts from nucleotide 403 and ends at 538, which results in the splitting of HBV surface and polymerase genes. Different HBV replication markers were evaluated for AAV-HBV1.04 plasmid–transfected cells, the AAV-HBV1.04 viral vector–transduced cells, and mice injected with the AAV-HBV1.04 viral vector.ResultsCompared with the previously reported AAV-HBV1.2 construct, direct transfection of AAV-HBV1.04 plasmid failed to produce hepatitis B surface antigen and progeny virus. Interestingly, AAV-HBV1.04 viral vector transduction could result in the formation of cccDNA and the production of all HBV replication markers in vitro and in vivo. The formation of cccDNA could be blocked by ATR (ataxia-telangiectasia and Rad3-related protein) inhibitors but not HBV reverse transcription inhibitor or capsid inhibitors. The AAV-HBV1.04 mouse supported long-term HBV replication and responded to antiviral treatments.ConclusionsThis AAV-HBV1.04 mouse model can support HBV cccDNA formation through ATR-mediated DNA damage response. The de novo formed cccDNA but not the parental AAV vector can lead to the production of hepatitis B surface antigen and HBV progeny. This model will provide a unique platform for studying HBV cccDNA and developing novel antivirals against HBV infection.

AURKA Regulates the Mechanism of ATR Mediated DNA Damage Response about Drug Resistance in Multiple Myeloma

Objective: Multiple myeloma (MM) is a cancer formed by malignant plasma cells. So far, drug resistance and relapse are major clinical problems in MM. Our previous studies showed that some chromosomal instability genes are involved in drug resistance, which AURKA expression is closely related survival. But the correlation between AURKA expression and clinical and how to participate in the mechanism of proliferation and drug resistance are unknown.Methods: Analyzing the Gene expression profiling (GEP) data and the relationship between AURKA expression and prognosis in MM. Bone marrow plasma cells from MM patients and health donor were collected by CD138. Detecting oncogene AURKA in presence of deletion and amplification by FISH. Detecting expression of mRNA by real-time qPCR. Immunofluorescence (IF) was used to detect the expression of protein. Comet assay was used to evaluate DNA repair. Then using the AURKA inhibitors and targeting AURKA, CCK-8 is used to detect the proliferation, WB detection of apoptosis, DNA Damage protein, IF detection of spindle formation and analyzing whether the AURKA is correlated with chromosome instability and drug resistance. In vivo and in vitro, combined with clinical medication bortezomib, Melphalan, lenalidomide. We detect apoptosis and DNA damage related protein and tumor volume in NOD/SCID mice. Changes in the culture supernatant of DNA damage response factor is used by real-time qPCR technique and ELASA detection of cell mass.Results: 1: Analyzing 350 patients with GEP data, the prognosis of patients with the AURKA high expression are better than the lowers (P=0). Patients with high expression of AURKA are mainly distributed in the high proliferation group. 2: Detecting the expression of mRNA by Real time qPCR technique, AURKA expression is significantly higher in newly diagnosed patients with 27.5% (n =11/n=40). The expression of AURKA protein is detected by IF assay. The results show that AURKA expression was higher in newly diagnosed (36.84%, n=14/n=38) and relapsed (50%, n=20/n=40) than that in normal donors. Patients with high expression of AURKA are closely related to the clinical relevance of anemia, bone destruction, high CR and high LDH. To further detect the relationship between AURKA expression and chromosome DNA copy number, the results show that AURKA copy number is normal in newly diagnosed patients (n=20). But there are 10% patients (n=2/n=20) with AURKA amplified in relapsed patients. 3: In vivo and in vitro, AURKA inhibitor combined with Bomtzomib, lenalidomide and Melphalan could enhance the sensitivity of BTZ and xenograft tumor biopsy showed reduced group AURKA, Ki67 expression. After over expression of AURKA, the proportion of cells with a single nucleus was decreased and the formation of multiple nuclei increased. Knocking down the AURKA, spindle formation is disorder. The expression of gamma-H2AX molecules and 53BP1 related DNA damage is abnormal. WB suggests that DNA damage is inhibited, phosphorylation of ATR, ATM, CHK2 reduction, phosphorylation of gamma-H2AX and BRCA increased. Further, real time qPCR technique and ELASA detection of DNA damage response factor in cell culture supernatant is mainly changed, such as IL-1 and IL6.Conclusion: High expression of AURKA is associated with poor prognosis in MM; Inhibition of AURKA can promote apoptosis, DNA damage and reduce the secretion of IL-6 that could enhance the sensitivity to bortezomib, lenalidomide and Melphalan. The abnormal expression of AURKA related spindle formation disorder and cell nuclear phenomena that cause chromosomal instability. AURKA is mainly involved in the drug resistance through the DNA damage response ATR-CHK1-gammaH2AX pathway. DisclosuresNo relevant conflicts of interest to declare.

NBS1 interacts with Notch signaling in neuronal homeostasis.

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom.

The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.

Topoisomerase II-binding protein 1 promotes the progression of prostate cancer via ATR-CHK1 signaling pathway.

DNA damage response (DDR) plays an important role in the progression of cancers, including prostate cancer (PCa). Topoisomerase II-binding protein 1 (TopBP1) is an essential promotor of ATR-mediated DDR. Herein, we investigated the association between TopBP1 and PCa and determined its effect on the progression of PCa. The expression and clinical features of TopBP1 were analyzed using large-scale cohort of tissue microarray analyses and The Cancer Genome Atlas database, which indicated that TopBP1 was positively correlated with high Gleason Score, advanced clinical and pathological stages, the metastasis status. Multivariate analysis revealed that the upregulation of TopBP1 was an independent predictor for a worse biochemical recurrence-free survival (BCR-free survival). Furthermore, we discovered that downregulation of TopBP1 significantly suppressed the growth and migration ability of PCa lines by loss-of-function assays in vitro. Further mechanistic investigations clarified that TopBP1 promoted proliferation and migration by activating ATR-Chk1 signaling pathway.

The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis.

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrestand elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to Chemotherapy by Abrogating the TopBP1/ATR-Mediated DNA Damage Response.

Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3β (GSK-3β) is an emerging target in human malignancies including PDAC.Experimental Design: Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence. 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage. These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.

ATR activated by EB virus facilitates chemotherapy resistance to cisplatin or 5-fluorouracil in human nasopharyngeal carcinoma.

PurposeEpstein–Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and increases the chemotherapy resistance of tumor cells. Although the mechanism by which EBV manipulates ataxia telangiectasia mutation (ATM)-mediated DNA damage response in NPC has been extensively studied, the relationship between ATR (ATM and Rad-3 related) and EBV infection is largely unexplored, and also the role of ATR in chemotherapy resistance in EBV-positive NPC has not been specifically reported.Materials and methodsLevels of γ-H2AX, latent membrane protein 1 (LMP1), and EBV-encoded RNA in clinical NPC and nasopharyngeal inflammation (NPI) specimens were examined using immunohistochemistry and in situ hybridization. The effects of EBV infection, chemotherapy drugs cisplatin (CDDP) and 5-fluorouracil (5-FU) treatment, and ATR silencing were assessed in NPC cells in vitro using immunofluorescence, Western blot, and flow cytometry.ResultsA notable increase of γ-H2AX expression was examined in the EBV-positive NPC clinical specimens. Additionally, we observed that the phosphorylation of ATR/checkpoint kinase 1 (CHK1) pathway protein was gradually activated along with the duration of EBV exposure in NPC cell lines, which was obviously inhibited after ATR depletion. Moreover, EBV infection promoted the resistance of NPC cells to CDDP and 5-FU, whereas the chemosensitivity of cells was significantly enhanced following ATR knockdown. Furthermore, ATR depletion caused both S-phase cell arrest and apoptosis, enhanced p53 phosphorylation, and impaired the formation of Rad51.ConclusionOur data suggest that EBV activation of ATR-mediated DNA damage response might result in chemotherapy resistance to CDDP and 5-FU in NPC. Accordingly, ATR knockdown may serve as an effective treatment strategy for chemotherapy-resistant, EBV-positive NPC.

TET3-mediated DNA oxidation promotes ATR-dependent DNA damage response.

An efficient, accurate, and timely DNA damage response (DDR) is crucial for the maintenance of genome integrity. Here, we report that ten-eleven translocation dioxygenase (TET) 3-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in response to ATR-dependent DDR regulates DNA repair. ATR-dependent DDR leads to dynamic changes in 5hmC levels and TET3 enzymatic activity. We show that TET3 is an ATR kinase target that oxidizes DNA during ATR-dependent DNA damage repair. Modulation of TET3 expression and activity affects DNA damage signaling and DNA repair and consequently cell death. Our results provide novel insight into ATR-mediated DDR, in which TET3-mediated DNA demethylation is crucial for efficient DNA repair and maintenance of genome stability.

Viral DNA replication-dependent DNA damage response activation during BK polyomavirus infection.

BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.

Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV LT-positive nuclear foci. Such a phenotype was recapitulated using our previously established system for visualizing MCPyV replication complexes in cells. By combining immunofluorescent staining, fluorescence in situ hybridization, and BrdU incorporation analysis, we demonstrate that DDR proteins are important for maintaining robust MCPyV DNA replication. This study not only provides the first look into the microscopic details of DDR factor/LT replication complexes at the MCPyV origin but also provides a platform for further studying the mechanistic role of host DDR factors in the MCPyV life cycle and virus-associated oncogenesis.

ATR suppresses apoptosis after UVB irradiation by controlling both translesion synthesis and alternative tolerance pathways.

Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase η (Pol η) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol η, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.

Viral protein R upregulates expression of ULBP2 on uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes

HIV-1 Vpr triggers NK cell-mediated lysis of infected cells by upregulating ULBP2, a ligand of the NKG2D receptor, through activation of the ATR-mediated DNA damage response. Herein, we demonstrate that Vpr augments ULBP2 expression on both infected and uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes. Indeed, the frequency of uninfected bystander cells expressing high levels of ULBP2 was elevated in a Vpr-dependent manner. Nevertheless, the same does not hold true for a Vpr mutant that is not packaged into virions, suggesting the involvement of virion-associated Vpr in this process. Additionally, we show that soluble Vpr has the ability to induce a DNA damage response and to augment cell-surface ULBP2 upon transducing target cells, including T cells, conditions known to promote NK cell-mediated killing. Overall, these findings suggest that Vpr could contribute to CD4+ T cell loss by rendering uninfected bystander cells susceptible to NK cell-mediated killing.

DNA damage response in microcephaly development of MCPH1 mouse model

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV–VI), while generate less late-born neurons that form a thinner outer layer (II–III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.