Cellular DNA Damage Response Research Articles

In Vivo Level of Poly(ADP-ribose)

PolyADP-ribosylation is a post-translational modification that plays key roles in cellular physiological functions and DNA damage responses. PolyADP-ribosylation is finely and dynamically regulated by various enzymes and factors involved in the synthesis and degradation of poly(ADP-ribose) (PAR). To better understand the function of polyADP-ribosylation, it is necessary to quantify and monitor the change of the in vivo level of PAR, the product of polyADP-ribosylation, which is rapidly turning over and kept in quite low level in cells or in organs. Recent developments of potent inhibitors of polyADP-ribosylation is expected to kill BRCA1/2-mutated breast cancer cells and ovarian cancer cells (synthetic lethality). To know the efficacy of these inhibitors in vivo, it is necessary to develop highly sensitive and reproducible methods to know PAR levels within cells or organs. However there have been several difficulties in measuring the physiologically low level of PAR without artefacts. Our experiments recently clarified that the method of sample preparation is very important in addition to the sensitivity and specificity. From reviewing the literature, including ours, we would like to emphasize the importance of the procedures of sample preparation for the assay, in addition to the sensitivity by comparing the reported PAR levels in vivo.

ERβ alters the chemosensitivity of luminal breast cancer cells by regulating p53 function.

Estrogen receptor α (ERα)-positive breast cancers tend to develop resistance to both endocrine therapy and chemotherapy. Despite recent progress in defining molecular pathways that confer endocrine resistance, the mechanisms that regulate chemotherapy response in luminal tumors remain largely elusive. Luminal tumors often express wild-type p53 that is a major determinant of the cellular DNA damage response. Similar to p53, the second ER subtype, ERβ, has been reported to inhibit breast tumorigenesis by acting alone or in collaboration with p53. However, a synergistic mechanism of action has not been described. Here, we suggest that ERβ relies on p53 to elicit its tumor repressive actions in ERα-positive breast cancer cells. Upregulation of ERβ and treatment with ERβ agonists potentiates the tumor suppressor function of p53 resulting in decreased survival. This effect requires molecular interaction between the two proteins that disrupts the inhibitory action of ERα on p53 leading to increased transcriptional activity of p53. In addition, we show that the same interaction alters the chemosensitivity of endocrine-resistant cells including their response to tamoxifen therapy. Our results suggest a collaboration of ERβ and p53 tumor suppressor activity in breast cancer cells that indicates the importance of ligand-regulated ERβ as a tool to target p53 activity and improve the clinical management of resistant disease.

Genome instability syndromes caused by impaired DNA repair and aberrant DNA damage responses.

Maintenance of genome integrity is essential for all organisms because genome information regulates cell proliferation, growth arrest, and vital metabolic processes in cells, tissues, organs, and organisms. Because genomes are constantly exposed to intrinsic and extrinsic genotoxic stress, cellular DNA repair machinery and proper DNA damage responses (DDR) have evolved to quickly eliminate genotoxic DNA lesions, thus maintaining the genome integrity suitably. In human, germline mutations in genes involved not only in cellular DNA repair pathways but also in cellular DDR machinery frequently predispose hereditary diseases associated with chromosome aberrations. These genetic syndromes typically displaying mutations in DNA repair/DDR-related genes are often called "genome instability syndromes." Common features of these hereditary syndromes include a high incidence of cancers and developmental abnormalities including short stature, microcephaly, and/or neurological deficiencies. However, precisely how impaired DNA repair and/or dysfunctional DDR pathologically promote(s) these syndromes are poorly understood. In this review article, we summarize the clinical symptoms of several representatives "genome instability syndromes" and propose the plausible pathogenesis thereof.

The ATM and Rad3-Related (ATR) Protein Kinase Pathway Is Activated by Herpes Simplex Virus 1 and Required for Efficient Viral Replication.

The ATM and Rad3-related (ATR) protein kinase and its downstream effector Chk1 are key sensors and organizers of the DNA damage response (DDR) to a variety of insults. Previous studies of herpes simplex virus 1 (HSV-1) showed no evidence for activation of the ATR pathway. Here we demonstrate that both Chk1 and ATR were phosphorylated by 3 h postinfection (h.p.i.). Activation of ATR and Chk1 was observed using 4 different HSV-1 strains in multiple cell types, while a specific ATR inhibitor blocked activation. Mechanistic studies point to early viral gene expression as a key trigger for ATR activation. Both pATR and pChk1 localized to the nucleus within viral replication centers, or associated with their periphery, by 3 h.p.i. Significant levels of pATR and pChk1 were also detected in the cytoplasm, where they colocalized with ICP4 and ICP0. Proximity ligation assays confirmed that pATR and pChk1 were closely and specifically associated with ICP4 and ICP0 in both the nucleus and cytoplasm by 3 h.p.i., but not with ICP8 or ICP27, presumably in a multiprotein complex. Chemically distinct ATR and Chk1 inhibitors blocked HSV-1 replication and infectious virion production, while inhibitors of ATM, Chk2, and DNA-dependent protein kinase (DNA-PK) did not. Together our data show that HSV-1 activates the ATR pathway at early stages of infection and that ATR and Chk1 kinase activities play important roles in HSV-1 replication fitness. These findings indicate that the ATR pathway may provide insight for therapeutic approaches.IMPORTANCE Viruses have evolved complex associations with cellular DNA damage response (DDR) pathways, which sense troublesome DNA structures formed during infection. The first evidence for activation of the ATR pathway by HSV-1 is presented. ATR is activated, and its downstream target Chk1 is robustly phosphorylated, during early stages of infection. Both activated proteins are found in the nucleus associated with viral replication compartments and in the cytoplasm associated with viral proteins. We also demonstrate that both ATR and Chk1 kinase activities are important for viral replication. The findings suggest that HSV-1 activates ATR and Chk1 during early stages of infection and utilizes the enzymes to promote its own replication. The observation may be exploitable for antiviral approaches.

Anti-apoptotic A1 is not essential for lymphoma development in E\xb5-Myc mice but helps sustain transplanted E\xb5-Myc tumour cells

The transcription factor c-MYC regulates a multiplicity of genes involved in cellular growth, proliferation, metabolism and DNA damage response and its overexpression is a hallmark of many tumours. Since MYC promotes apoptosis under conditions of stress, such as limited availability of nutrients or cytokines, MYC-driven cells are very much dependent on signals that inhibit cell death. Stress signals trigger apoptosis via the pathway regulated by opposing fractions of the BCL-2 protein family and previous genetic studies have shown that the development of B lymphoid tumours in Eµ-Myc mice is critically dependent on expression of pro-survival BCL-2 relatives MCL-1, BCL-W and, to a lesser extent, BCL-XL, but not BCL-2 itself, and that sustained growth of these lymphomas is dependent on MCL-1. Using recently developed mice that lack expression of all three functional pro-survival A1 genes, we show here that the kinetics of lymphoma development in Eµ-Myc mice and the competitive repopulation capacity of Eµ-Myc haemopoietic stem and progenitor cells is unaffected by the absence of A1. However, conditional loss of a single remaining functional A1 gene from transplanted A1-a−/−A1-bfl/flA1-c−/− Eµ-Myc lymphomas slowed their expansion, significantly extending the life of the transplant recipients. Thus, A1 contributes to the survival of malignant Eµ-Myc-driven B lymphoid cells. These results strengthen the case for BFL-1, the human homologue of A1, being a valid target for drug development for MYC-driven tumours.

ATM directs DNA damage responses and proteostasis via genetically separable pathways.

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. We genetically separated the activation of ATM by DNA damage from that by oxidative stress using separation-of-function mutations. We found that deficient activation of ATM by the Mre11-Rad50-Nbs1 complex and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of a variant ATM incapable of activation by oxidative stress resulted in widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicate ATM in the control of protein homeostasis.

DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.

Notch1-Cyclin E-p27kip1 and RAD17 form a network of proteins which control cellular proliferation and DNA damage response in cholangiocarcinoma

Notch1-Cyclin E-p27kip1 and RAD17 form a network of proteins which control cellular proliferation and DNA damage response in cholangiocarcinoma

Abstract A124: Discovery of the clinical candidate AZD1390: a high-quality, potent, and selective inhibitor of ATM kinase with the ability to cross the blood-brain barrier

Abstract Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor, and current treatment (surgery followed by fractionated radiotherapy and temozolomide) provides a median survival of just 12-15 months (1,2). The poor prognosis associated with GBM is attributed to an extensive infiltration into surrounding brain tissue (thereby limiting the effectiveness of surgical excision), an intrinsic chemo/radioresistance of the tumor, and the presence of the blood-brain barrier (BBB), which limits the ability of certain chemotherapies to reach the tumor. Ataxia telangiectasia mutant (ATM) is a serine/threonine protein kinase from the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases and plays a crucial role in the cellular DNA damage response signalling activated by DNA double-strand breaks (DSB). Activated ATM promotes DNA repair and S/G1-cell cycle checkpoints to prevent premature mitosis, maintain genomic integrity, and promote appropriate cell survival or death pathways. DSBs arise intrinsically through the collapse of stalled replication forks, which are induced by a wide range of chemotherapies, or extrinsically through exposure to ionizing radiation. Therefore, ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitize tumors to chemo/radiotherapy. The optimization of compound properties suitable to allow efficient BBB penetration remains a significant challenge within Medicinal Chemistry, and failure to consider these can severely restrict the utility of an agent for CNS disease. Herein, we describe the identification of AZD1390, a first-in-class orally available and CNS penetrant ATM inhibitor suitable for the treatment of intracranial malignancies. This presentation represents the first disclosure of the Medicinal Chemistry strategies employed to optimize BBB penetration, alongside the SAR for ATM potency, selectivity, and pharmacokinetic properties. AZD1390 is an exceptionally potent inhibitor of ATM in cells (IC50 = 0.78 nM) with >10,000-fold selectivity over closely related members of the PIKK family of enzymes and excellent selectivity across a broad panel of kinases. AZD1390 displays excellent oral bioavailability in preclinical species (66% in rat and 74% in dog), is not a substrate for human efflux transporters, and has been shown to efficiently cross the BBB in non-human primate PET studies. Profound tumor regressions and increased animal survival (>50 days) have been observed in orthotopic xenograft models of brain cancer following just 2 or 4 days combination treatment of AZD1390 with radiotherapy, compared to radiotherapy treatment alone. These data support the potential of CNS-penetrant ATM inhibitors to provide an important new therapeutic agent for the treatment of intracranial malignancies. AZD1390 is currently undergoing early clinical assessment.

Abstract A112: RanBP9 protects cells from genotoxic stress and increased expression is predictive of worse response to platinum in NSCLC patients

Abstract Platinum-based therapies currently represent the most common first-line treatment for non-small cell lung cancer (NSCLC). The efficacy of platinum compounds depends on the activation of the cellular DNA damage response (DDR), which leads to cancer cell death. However, severe toxic effects and development of resistance represent major limitations in the use of these drugs. Thus, a better mechanistic understanding and new prognostic and predictive biomarkers of the DDR are required to improve outcomes of NSCLC treatment. We have previously reported that RanBP9 (Palmieri et al., 2016), a poorly characterized scaffold protein, participates in the DDR and that its downregulation causes enhanced sensitivity to DNA damage induced by ionizing radiation. In the present study, we used immunohistochemical analysis to reveal that RanBP9 expression is significantly and commonly elevated in 178 lung tumors of different histotypes compared to their normal adjacent tissue (p<0.02 - 0.001). We also show that knockout (KO) of RanBP9 in A549 NSCLC cell lines resulted in reduced DDR and higher levels of cisplatin-induced apoptosis both in vitro and in vivo. These findings were validated by a retrospective analysis of 134 NSCLC patients in which higher levels of RanBP9 associated with tumor stage (p<0.0001), and poor response to platinum compounds as first-line treatment (PFS, HR (RanBP9 positive vs negative) 1.71, 95% CI 1.142 - 2.563, p = 0.0093). Finally, we show that ablation of RanBP9 is associated with overactivation of poly(ADP-ribose) polymerase (PARP) and increased cisplatin antineoplastic efficacy in combination with PARP inhibitors, indicating that the absence of RanBP9 results in a BRCAness-like phenotype. Overall, these results reveal that RanBP9 is a novel predictive biomarker of response to genotoxic treatments in NSCLC, paving the way to prospective studies aimed at assessing its clinical prognostic value and therapeutic targeting. Citation Format: Anna Tessari, Kareesma Parbhoo, Meghan Pawlikowski, Matteo Fassan, Eliana Rulli, Claudia Foray, Alessandra Fabbri, Valerio Embrione, Monica Ganzinelli, Marina Capece, Moray J. Campbell, Massimo Broggini, Krista La Perle, Gabriella Farina, Sara Cole, Mirko Marabese, Joseph M. Amann, David P. Carbone, Marina C. Garassino, Carlo M. Croce, Dario Palmieri, Vincenzo Coppola. RanBP9 protects cells from genotoxic stress and increased expression is predictive of worse response to platinum in NSCLC patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A112.

Complex DNA Damage Induced by High Linear Energy Transfer Alpha-Particles and Protons Triggers a Specific Cellular DNA Damage Response

PurposeTo investigate the precise mechanism of recognition and processing of ionizing radiation (IR)-induced complex DNA damage (CDD), where two or more DNA lesions are in close proximity, in cellular DNA which is packaged with histones to form chromatin.Methods and MaterialsHeLa and oropharyngeal squamous cell carcinoma (UMSCC74A and UMSCC6) cells were irradiated with high linear energy transfer (LET) α-particles or protons, versus low-LET protons and X rays. At various time points after irradiation, site-specific histone post-translational modifications were analyzed by quantitative Western blotting; DNA damage and repair were measured by different versions of the comet assay; and cell survival was determined using clonogenic assays.ResultsSite-specific histone post-translational modifications after low- and high-LET radiation, particularly proton irradiation, were screened, aiming to identify those responsive to CDD. We demonstrate that histone H2B ubiquitylated on lysine 120 (H2Bub) is specifically induced several hours after irradiation in response to high-LET α-particles and protons but not by low-LET protons or X rays/γ-radiation. This is associated with increased levels of CDD, which contributes to decreased cell survival. We further discovered that modulation of H2Bub is under the control of two E3 ubiquitin ligases, MSL2 and RNF20/RNF40 complex, whose depletion leads to defective processing and further persistence of CDD, and to additional decreased cell survival after irradiation.ConclusionThis study demonstrates that the signaling and repair of CDD, particularly induced by high-LET IR is co-ordinated through the specific induction of H2Bub catalyzed by MSL2 and RNF20/40, a mechanism that contributes significantly to cell survival after irradiation.

The RNA processing factors THRAP3 and BCLAF1 promote the DNA damage response through selective mRNA splicing and nuclear export.

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.

Protoparvovirus Interactions with the Cellular DNA Damage Response.

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.

Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

ABSTRACTDouble-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication.IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.

Adenovirus Core Protein VII Downregulates the DNA Damage Response on the Host Genome.

Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier to successful infection. The adenovirus genome is packaged with protein VII, a virally encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by the cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (γH2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in γH2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to abrogate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin.IMPORTANCE The DNA damage response (DDR) is a cellular network that is crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that can cause a multitude of diseases, such as respiratory infections and conjunctivitis. Here we describe how a small adenovirus core protein that localizes to host chromatin during infection can globally downregulate the DDR. Our study focuses on key players in the damage signaling pathway and highlights how viral manipulation of chromatin may influence access of DDR proteins to the host genome.

Identification of ATIC as a Novel Target for Chemoradiosensitization

Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

Chronic polycyclic aromatic hydrocarbon exposure causes DNA damage and genomic instability in lung epithelial cells.

Cell exposure to atmospheric polycyclic aromatic hydrocarbons (PAHs) is closely associated with DNA damage and genomic instability. We assessed the mechanisms of chronic and acute PAH exposure-induced genotoxicity in two human lung epithelial cell lines, A549 and NC-H1975. We sampled atmospheric PAHs at the Xixi Campus, Zhejiang University in Hangzhou, China during August (non-haze episode) and November (haze episode) 2015. We identified vehicle emissions as a dominant anthropogenic PAH source in our study. PAHs were extracted according to the United States Environmental Protection Agency Method TO-13A. We found that chronic PAH exposure saturated lung cell xenobiotic metabolic pathways, promoting intercellular reactive oxygen species production and accumulation. Chronic alteration of the cellular redox status resulted in DNA damage and genomic instability. Chronic PAH exposure also perturbed the cellular DNA damage response, inducing S phase arrest and inhibiting apoptosis. Dysregulation of PAH metabolism and the DNA damage response altered cellular homeostasis and increased cell susceptibility to subsequent PAH exposures, thereby enhancing the likelihood of genomic mutation and instability.

Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex

We observed extra-telomeric binding of the telomere repeat binding factor TRF2 within the promoter of the cyclin-dependent kinase CDKNIA (p21/CIP1/WAF1). This result in TRF2 induced transcription repression of p21. Interestingly, p21 repression was through engagement of the REST-coREST-LSD1-repressor complex and altered histone marks at the p21 promoter in a TRF2-dependent fashion. Furthermore, mutational analysis shows p21 repression requires interaction of TRF2 with a p21 promoter G-quadruplex. Physiologically, TRF2-mediated p21 repression attenuated drug-induced activation of cellular DNA damage response by evading G2/M arrest in cancer cells. Together these reveal for the first time role of TRF2 in REST- repressor complex mediated transcription repression.

Integration of the TGx-28.65 genomic biomarker with the flow cytometry micronucleus test to assess the genotoxicity of disperse orange and 1,2,4-benzenetriol in human TK6 cells

In vitro gene expression signatures to predict toxicological responses can provide mechanistic context for regulatory testing. We previously developed the TGx-28.65 genomic biomarker from a database of gene expression profiles derived from human TK6 cells exposed to 28 well-known compounds. The biomarker comprises 65 genes that can classify chemicals as DNA damaging or non-DNA damaging. In this study, we applied the TGx-28.65 genomic biomarker in parallel with the in vitro micronucleus (MN) assay to determine if two chemicals of regulatory interest at Health Canada, disperse orange (DO: the orange azo dye 3-[[4-[(4-Nitrophenyl)azo]phenyl] benzylamino]propanenitrile) and 1,2,4-benzenetriol (BT: a metabolite of benzene) are genotoxic or non-genotoxic. Both chemicals caused dose-dependent declines in relative survival and increases in apoptosis. A strong significant increase in MN induction was observed for all concentrations of BT; the top two concentrations of DO also caused a statistically significant increase in MN, but these increases were <2-fold above controls. TGx-28.65 analysis classified BT as genotoxic at all three concentrations and DO as genotoxic at the mid and high concentrations. Thus, although DO only caused a small increase in MN, this response was sufficient to induce a cellular DNA damage response. Benchmark dose modeling confirmed that BT is much more potent than DO. The results strongly suggest that follow-up work is required to assess whether DO and BT are also genotoxic in vivo. This is particularly important for DO, which may require metabolic activation by bacterial gut flora to fully induce its genotoxic potential. Our previously published data and this proof of concept study suggest that the TGx-28.65 genomic biomarker has the potential to add significant value to existing approaches used to assess genotoxicity.

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication.IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production.