Browsing by Subject "DNA Damage"
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Item Open Access A genome-wide RNAi screen reveals multiple regulators of caspase activation.(The Journal of cell biology, 2007-11-12) Yi, Caroline H; Sogah, Dodzie K; Boyce, Michael; Degterev, Alexei; Christofferson, Dana E; Yuan, JunyingApoptosis is an evolutionally conserved cellular suicide mechanism that can be activated in response to a variety of stressful stimuli. Increasing evidence suggests that apoptotic regulation relies on specialized cell death signaling pathways and also integrates diverse signals from additional regulatory circuits, including those of cellular homeostasis. We present a genome-wide RNA interference screen to systematically identify regulators of apoptosis induced by DNA damage in Drosophila melanogaster cells. We identify 47 double- stranded RNA that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. Our data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death.Item Open Access A recessive variant of XRCC4 predisposes to non- BRCA1/2 breast cancer in chinese women and impairs the DNA damage response via dysregulated nuclear localization.(Oncotarget, 2014-12) He, Min; Hu, Xin; Chen, Li; Cao, A-Yong; Yu, Ke-Da; Shi, Ting-Yan; Kuang, Xia-Ying; Shi, Wen-Biao; Ling, Hong; Li, Shan; Qiao, Feng; Yao, Ling; Wei, Qingyi; Di, Gen-Hong; Shao, Zhi-MingXRCC4 plays a crucial role in the non-homologous end joining pathway that maintains genome stability. In this two-stage case-control study with 1,764 non-BRCA1/2 breast cancer patients and 1,623 cancer-free controls, we investigated the contribution of genetic variants of XRCC4 to breast cancer susceptibility in Chinese women. We identified a recessive missense variant, rs3734091 (c.739G>T, p.Ala247Ser), of XRCC4 that was significantly associated with an increased risk of breast cancer (odds ratio [OR] = 3.92, P = 0.007), particularly with the risk of developing triple-negative breast cancer (OR = 18.65, P < 0.0001). This p.Ala247Ser variant disturbed the nuclear localization of XRCC4 in cells homozygous for the rs3734091-T allele but not in heterozygous cells at both the cellular and tissue levels. In heterozygous cells, wild-type XRCC4 facilitated the nuclear localization of the XRCC4A247S mutant, thus compensating for the impaired localization of XRCC4A247S. This provided a biological mechanism by which rs3734091 conferred an increased susceptibility to non-BRCA1/2 breast cancer exclusively under a recessive model. Further functional analyses revealed that p.Ala247Ser impaired the DNA damage repair capacity and ultimately perturbed genomic stability. Taken together, our findings document the role of XRCC4 in non-BRCA1/2 breast cancer predisposition and reveal its underlying biological mechanism of action.Item Open Access Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: cellular model of pathology.(PLoS Genet, 2010-01-15) Haugen, Astrid C; Di Prospero, Nicholas A; Parker, Joel S; Fannin, Rick D; Chou, Jeff; Meyer, Joel N; Halweg, Christopher; Collins, Jennifer B; Durr, Alexandra; Fischbeck, Kenneth; Van Houten, BennettThe neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials.Item Restricted Association between DNA damage response and repair genes and risk of invasive serous ovarian cancer.(PLoS One, 2010-04-08) Schildkraut, Joellen M; Iversen, Edwin S; Wilson, Melanie A; Clyde, Merlise A; Moorman, Patricia G; Palmieri, Rachel T; Whitaker, Regina; Bentley, Rex C; Marks, Jeffrey R; Berchuck, AndrewBACKGROUND: We analyzed the association between 53 genes related to DNA repair and p53-mediated damage response and serous ovarian cancer risk using case-control data from the North Carolina Ovarian Cancer Study (NCOCS), a population-based, case-control study. METHODS/PRINCIPAL FINDINGS: The analysis was restricted to 364 invasive serous ovarian cancer cases and 761 controls of white, non-Hispanic race. Statistical analysis was two staged: a screen using marginal Bayes factors (BFs) for 484 SNPs and a modeling stage in which we calculated multivariate adjusted posterior probabilities of association for 77 SNPs that passed the screen. These probabilities were conditional on subject age at diagnosis/interview, batch, a DNA quality metric and genotypes of other SNPs and allowed for uncertainty in the genetic parameterizations of the SNPs and number of associated SNPs. Six SNPs had Bayes factors greater than 10 in favor of an association with invasive serous ovarian cancer. These included rs5762746 (median OR(odds ratio)(per allele) = 0.66; 95% credible interval (CI) = 0.44-1.00) and rs6005835 (median OR(per allele) = 0.69; 95% CI = 0.53-0.91) in CHEK2, rs2078486 (median OR(per allele) = 1.65; 95% CI = 1.21-2.25) and rs12951053 (median OR(per allele) = 1.65; 95% CI = 1.20-2.26) in TP53, rs411697 (median OR (rare homozygote) = 0.53; 95% CI = 0.35 - 0.79) in BACH1 and rs10131 (median OR( rare homozygote) = not estimable) in LIG4. The six most highly associated SNPs are either predicted to be functionally significant or are in LD with such a variant. The variants in TP53 were confirmed to be associated in a large follow-up study. CONCLUSIONS/SIGNIFICANCE: Based on our findings, further follow-up of the DNA repair and response pathways in a larger dataset is warranted to confirm these results.Item Open Access BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation.(Cell reports, 2020-09) Edwards, Drake S; Maganti, Rohin; Tanksley, Jarred P; Luo, Jie; Park, James JH; Balkanska-Sinclair, Elena; Ling, Jinjie; Floyd, Scott REffective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF.Item Open Access CometChip enables parallel analysis of multiple DNA repair activities.(DNA repair, 2021-10) Ge, Jing; Ngo, Le P; Kaushal, Simran; Tay, Ian J; Thadhani, Elina; Kay, Jennifer E; Mazzucato, Patrizia; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Sobol, Robert W; Samson, Leona D; Floyd, Scott R; Engelward, Bevin PDNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.Item Open Access DNA adducts of decarbamoyl mitomycin C efficiently kill cells without wild-type p53 resulting from proteasome-mediated degradation of checkpoint protein 1.(Chem Res Toxicol, 2010-07-19) Boamah, Ernest K; Brekman, Angelika; Tomasz, Maria; Myeku, Natura; Figueiredo-Pereira, Maria; Hunter, Senyene; Meyer, Joel; Bhosle, Rahul C; Bargonetti, JillThe mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-beta-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-beta stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.Item Open Access Gene expression signatures that predict radiation exposure in mice and humans.(PLoS Med, 2007-04) Dressman, Holly K; Muramoto, Garrett G; Chao, Nelson J; Meadows, Sarah; Marshall, Dawn; Ginsburg, Geoffrey S; Nevins, Joseph R; Chute, John PBACKGROUND: The capacity to assess environmental inputs to biological phenotypes is limited by methods that can accurately and quantitatively measure these contributions. One such example can be seen in the context of exposure to ionizing radiation. METHODS AND FINDINGS: We have made use of gene expression analysis of peripheral blood (PB) mononuclear cells to develop expression profiles that accurately reflect prior radiation exposure. We demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, ranging from 50 cGy to 1,000 cGy. Likewise, a molecular signature of radiation response developed solely from irradiated human patient samples can predict and distinguish irradiated human PB samples from nonirradiated samples with an accuracy of 90%, sensitivity of 85%, and specificity of 94%. We further demonstrate that a radiation profile developed in the mouse can correctly distinguish PB samples from irradiated and nonirradiated human patients with an accuracy of 77%, sensitivity of 82%, and specificity of 75%. Taken together, these data demonstrate that molecular profiles can be generated that are highly predictive of different levels of radiation exposure in mice and humans. CONCLUSIONS: We suggest that this approach, with additional refinement, could provide a method to assess the effects of various environmental inputs into biological phenotypes as well as providing a more practical application of a rapid molecular screening test for the diagnosis of radiation exposure.Item Open Access HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53.(Genes Dev, 2007-04-01) Sasaki, Toru; Gan, Eugene C; Wakeham, Andrew; Kornbluth, Sally; Mak, Tak W; Okada, HitoshiIn response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. Here, we describe an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21. Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53-p300 complex formation and decreased p53 acetylation. Furthermore, consistent with our in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. Our data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53.Item Open Access Human genetic risk of treatment with antiviral nucleoside analog drugs that induce lethal mutagenesis: The special case of molnupiravir.(Environmental and molecular mutagenesis, 2022-01) Waters, Michael D; Warren, Stafford; Hughes, Claude; Lewis, Philip; Zhang, FengyuThis review considers antiviral nucleoside analog drugs, including ribavirin, favipiravir, and molnupiravir, which induce genome error catastrophe in SARS-CoV or SARS-CoV-2 via lethal mutagenesis as a mode of action. In vitro data indicate that molnupiravir may be 100 times more potent as an antiviral agent than ribavirin or favipiravir. Molnupiravir has recently demonstrated efficacy in a phase 3 clinical trial. Because of its anticipated global use, its relative potency, and the reported in vitro "host" cell mutagenicity of its active principle, β-d-N4-hydroxycytidine, we have reviewed the development of molnupiravir and its genotoxicity safety evaluation, as well as the genotoxicity profiles of three congeners, that is, ribavirin, favipiravir, and 5-(2-chloroethyl)-2'-deoxyuridine. We consider the potential genetic risks of molnupiravir on the basis of all available information and focus on the need for additional human genotoxicity data and follow-up in patients treated with molnupiravir and similar drugs. Such human data are especially relevant for antiviral NAs that have the potential of permanently modifying the genomes of treated patients and/or causing human teratogenicity or embryotoxicity. We conclude that the results of preclinical genotoxicity studies and phase 1 human clinical safety, tolerability, and pharmacokinetics are critical components of drug safety assessments and sentinels of unanticipated adverse health effects. We provide our rationale for performing more thorough genotoxicity testing prior to and within phase 1 clinical trials, including human PIG-A and error corrected next generation sequencing (duplex sequencing) studies in DNA and mitochondrial DNA of patients treated with antiviral NAs that induce genome error catastrophe via lethal mutagenesis.Item Open Access Human longevity and variation in GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidant pathway genes: cross sectional and longitudinal studies.(Exp Gerontol, 2012-05) Soerensen, Mette; Dato, Serena; Tan, Qihua; Thinggaard, Mikael; Kleindorp, Rabea; Beekman, Marian; Jacobsen, Rune; Suchiman, H Eka D; de Craen, Anton JM; Westendorp, Rudi GJ; Schreiber, Stefan; Stevnsner, Tinna; Bohr, Vilhelm A; Slagboom, P Eline; Nebel, Almut; Vaupel, James W; Christensen, Kaare; McGue, Matt; Christiansen, LeneHere we explore association with human longevity of common genetic variation in three major candidate pathways: GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidants by investigating 1273 tagging SNPs in 148 genes composing these pathways. In a case-control study of 1089 oldest-old (age 92-93) and 736 middle-aged Danes we found 1 pro/antioxidant SNP (rs1002149 (GSR)), 5 GH/IGF-1/INS SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs3842755 (INS), rs572169 (GHSR), rs9456497 (IGF2R)) and 5 DNA repair SNPs (rs11571461 (RAD52), rs13251813 (WRN), rs1805329 (RAD23B), rs2953983 (POLB), rs3211994 (NTLH1)) to be associated with longevity after correction for multiple testing. In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes we found 2 pro/antioxidant SNPs (rs10047589 (TNXRD1), rs207444 (XDH)), 1 GH/IGF-1/INS SNP (rs26802 (GHRL)) and 3 DNA repair SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing. When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated the same directions of effect (p<0.05), while rs9456497 (IGF2R) and rs1157146 (RAD52) showed non-significant tendencies, indicative of effects also in late life survival. In addition, rs207444 (XDH) presented the same direction of effect when inspecting the 6 SNPs from the longitudinal study in the case-control data, hence, suggesting an effect also in survival from middle age to old age. No formal replications were observed when investigating the 11 SNPs from the case-control study in 1613 oldest-old (age 95-110) and 1104 middle-aged Germans, although rs11571461 (RAD52) did show a supportive non-significant tendency (OR=1.162, 95% CI=0.927-1.457). The same was true for rs10047589 (TNXRD1) (HR=0.758, 95%CI=0.543-1.058) when examining the 6 SNPs from the longitudinal study in a Dutch longitudinal cohort of oldest-old (age 85+, N=563). In conclusion, the present candidate gene based association study, the largest to date applying a pathway approach, not only points to potential new longevity loci, but also underlines the difficulties of replicating association findings in independent study populations and thus the difficulties in identifying universal longevity polymorphisms.Item Open Access Mitochondrial DNA damage induced autophagy, cell death, and disease.(Front Biosci (Landmark Ed), 2016-01-01) Van Houten, Bennett; Hunter, Senyene E; Meyer, Joel NMammalian mitochondria contain multiple small genomes. While these organelles have efficient base excision removal of oxidative DNA lesions and alkylation damage, many DNA repair systems that work on nuclear DNA damage are not active in mitochondria. What is the fate of DNA damage in the mitochondria that cannot be repaired or that overwhelms the repair system? Some forms of mitochondrial DNA damage can apparently trigger mitochondrial DNA destruction, either via direct degradation or through specific forms of autophagy, such as mitophagy. However, accumulation of certain types of mitochondrial damage, in the absence of DNA ligase III (Lig3) or exonuclease G (EXOG), can directly trigger cell death. This review examines the cellular effects of persistent damage to mitochondrial genomes and discusses the very different cell fates that occur in response to different kinds of damage.Item Open Access Nrf2 inactivation enhances placental angiogenesis in a preeclampsia mouse model and improves maternal and fetal outcomes.(Science signaling, 2017-05-16) Nezu, Masahiro; Souma, Tomokazu; Yu, Lei; Sekine, Hiroki; Takahashi, Nobuyuki; Wei, Andrew Zu-Sern; Ito, Sadayoshi; Fukamizu, Akiyoshi; Zsengeller, Zsuzsanna K; Nakamura, Tomohiro; Hozawa, Atsushi; Karumanchi, S Ananth; Suzuki, Norio; Yamamoto, MasayukiPlacental activation of the renin-angiotensin system (RAS) plays a key role in the pathogenesis of preeclampsia. Reactive oxygen species (ROS) are thought to affect placental angiogenesis, which is critical for preventing preeclampsia pathology. We examined the role of ROS in preeclampsia by genetically modifying the Keap1-Nrf2 pathway, a cellular antioxidant defense system, in a mouse model of RAS-induced preeclampsia. Nrf2 deficiency would be expected to impair cellular antioxidant responses; however, Nrf2 deficiency in preeclamptic mice improved maternal and fetal survival, ameliorated intra-uterine growth retardation, and augmented oxidative DNA damage. Furthermore, the placentas of Nrf2-deficient mice had increased endothelial cell proliferation with dense vascular networks. In contrast, the placentas of preeclamptic mice with overactive Nrf2 showed repressed angiogenesis, which was associated with decreased expression of genes encoding angiogenic chemokines and cytokines. Our findings support the notion that ROS-mediated signaling is essential for maintaining placental angiogenesis in preeclampsia and may provide mechanistic insight into the negative results of clinical trials for antioxidants in preeclampsia.Item Open Access Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.(PLoS One, 2014) Stimpson, Kaitlin M; Sullivan, Lori L; Kuo, Molly E; Sullivan, Beth AThe short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA). The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2) causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.Item Open Access PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.(Curr Protoc Toxicol, 2016-02-01) Gonzalez-Hunt, Claudia P; Rooney, John P; Ryde, Ian T; Anbalagan, Charumathi; Joglekar, Rashmi; Meyer, Joel NBecause of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.Item Open Access Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors.(Cell Cycle, 2013-03-15) Weingeist, David M; Ge, Jing; Wood, David K; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A; Yaffe, Michael B; Floyd, Scott; Engelward, Bevin PA key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.Item Open Access Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase.(Cell reports, 2015-06-18) Choy, Meng S; Yusoff, Permeen; Lee, Irene C; Newton, Jocelyn C; Goh, Catherine W; Page, Rebecca; Shenolikar, Shirish; Peti, WolfgangThe attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.Item Open Access Suppression of CHK1 by ETS Family Members Promotes DNA Damage Response Bypass and Tumorigenesis.(Cancer discovery, 2015-05) Lunardi, Andrea; Varmeh, Shohreh; Chen, Ming; Taulli, Riccardo; Guarnerio, Jlenia; Ala, Ugo; Seitzer, Nina; Ishikawa, Tomoki; Carver, Brett S; Hobbs, Robin M; Quarantotti, Valentina; Ng, Christopher; Berger, Alice H; Nardella, Caterina; Poliseno, Laura; Montironi, Rodolfo; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Signoretti, Sabina; Pandolfi, Pier PaoloUNLABELLED:The ETS family of transcription factors has been repeatedly implicated in tumorigenesis. In prostate cancer, ETS family members, such as ERG, ETV1, ETV4, and ETV5, are frequently overexpressed due to chromosomal translocations, but the molecular mechanisms by which they promote prostate tumorigenesis remain largely undefined. Here, we show that ETS family members, such as ERG and ETV1, directly repress the expression of the checkpoint kinase 1 (CHK1), a key DNA damage response cell-cycle regulator essential for the maintenance of genome integrity. Critically, we find that ERG expression correlates with CHK1 downregulation in human patients and demonstrate that Chk1 heterozygosity promotes the progression of high-grade prostatic intraepithelial neoplasia into prostatic invasive carcinoma in Pten(+) (/-) mice. Importantly, CHK1 downregulation sensitizes prostate tumor cells to etoposide but not to docetaxel treatment. Thus, we identify CHK1 as a key functional target of the ETS proto-oncogenic family with important therapeutic implications. SIGNIFICANCE:Genetic translocation and aberrant expression of ETS family members is a common event in different types of human tumors. Here, we show that through the transcriptional repression of CHK1, ETS factors may favor DNA damage accumulation and consequent genetic instability in proliferating cells. Importantly, our findings provide a rationale for testing DNA replication inhibitor agents in ETS-positive TP53-proficient tumors.Item Open Access The bromodomain protein Brd4 insulates chromatin from DNA damage signalling.(Nature, 2013-06-13) Floyd, Scott R; Pacold, Michael E; Huang, Qiuying; Clarke, Scott M; Lam, Fred C; Cannell, Ian G; Bryson, Bryan D; Rameseder, Jonathan; Lee, Michael J; Blake, Emily J; Fydrych, Anna; Ho, Richard; Greenberger, Benjamin A; Chen, Grace C; Maffa, Amanda; Del Rosario, Amanda M; Root, David E; Carpenter, Anne E; Hahn, William C; Sabatini, David M; Chen, Clark C; White, Forest M; Bradner, James E; Yaffe, Michael BDNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.Item Open Access The oncoprotein DEK affects the outcome of PARP1/2 inhibition during mild replication stress.(PloS one, 2019-01) Ganz, Magdalena; Vogel, Christopher; Czada, Christina; Jörke, Vera; Gwosch, Eva Christina; Kleiner, Rebecca; Pierzynska-Mach, Agnieszka; Zanacchi, Francesca Cella; Diaspro, Alberto; Kappes, Ferdinand; Bürkle, Alexander; Ferrando-May, ElisaDNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.