Cidofovir is active against human papillomavirus positive and negative head and neck and cervical tumor cells by causing DNA damage as one of its working mechanisms.

PubMed

Mertens, Barbara; Nogueira, Tatiane; Stranska, Ruzena; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert

2016-07-26

Human papillomavirus (HPV) causes cervical cancer and a large fraction of head and neck squamous cell carcinomas (HNSCC). Cidofovir (CDV) proved efficacious in the treatment of several HPV-induced benign and malignant hyper proliferations. To provide a better insight into how CDV selectively eradicates transformed cells, HPV+ and HPV- cervical carcinoma and HNSCC cell lines were compared to normal cells for antiproliferative effects, CDV metabolism, drug incorporation into cellular DNA, and DNA damage. Incorporation of CDV into cellular DNA was higher in tumor cells than in normal cells and correlated with CDV antiproliferative effects, which were independent of HPV status. Increase in phospho-ATM levels was detected following CDV exposure and higher levels of γ-H2AX (a quantitative marker of double-strand breaks) were measured in tumor cells compared to normal cells. A correlation between DNA damage and CDV incorporation into DNA was found but not between DNA damage and CDV antiproliferative effects. These data indicate that CDV antiproliferative effects result from incorporation of the drug into DNA causing DNA damage. However, the anti-tumor effects of CDV cannot be exclusively ascribed to DNA damage. Furthermore, CDV can be considered a promising broad spectrum anti-cancer agent, not restricted to HPV+ lesions.

[Combi-molecules: a global approach towards better chemoselectivity and chemosensitivity].

PubMed

Matheson, Stéphanie; Qiu, Qiyu; Brahimi, Fouad; Dudouit, Fabienne; Banerjee, Ranjita; Rachid, Zakaria; Jean-Claude, Bertrand J

2004-12-01

It is now known that tumour cells possess many signaling pathways to repair damage inflicted by alkylating agents. However, most of these cytotoxic agents only target DNA and this does not suffice to induce sustained antiproliferative activity. Furthermore, the efficacy of antitumour alkylating agents is hampered by a lack of selectivity for tumour tissues. To circumvent these problems, we recently designed a novel strategy termed combi-targeting that sought to synthesize compounds capable of not only damaging DNA, but also blocking signaling associated with aggressive proliferation. The first prototypes described herein can block signaling associated with the epidermal growth factor receptor (EGFR) and significantly damage DNA. In addition to their binary EGFR/DNA targeting properties, we demonstrated that their effects are selective for cells to which EGFR has conferred a proliferative advantage. These novel agents with mixed targeting properties are termed "combi-molecules".

Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.

PubMed

Kitamoto, Sachiko; Matsuyama, Ryoko; Uematsu, Yasuaki; Ogata, Keiko; Ota, Mika; Yamada, Toru; Miyata, Kaori; Funabashi, Hitoshi; Saito, Koichi

2015-07-01

The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of red light irradiation on spermatozoa DNA

NASA Astrophysics Data System (ADS)

Chow, Kay W.; Preece, Daryl; Gomez-Godinez, Veronica; Berns, Michael W.

2016-09-01

A key goal in the conservation of endangered species is to increase successful reproduction. In cases where traditional methods of in vitro fertilization are unsuccessful, new methods of assisted reproduction are needed. One option is selective fertilization via optically trapped sperm. A more passive option is red light irradiation. Red light irradiation has been shown to increase sperm motility, thus increasing fertilizing potential. However, there is some concern that exposure to laser irradiation induces the production of oxidative species in cells, which can be damaging to DNA. In order to test the safety of irradiating sperm, sperm samples were exposed to 633 nm laser light and their DNA were tested for oxidative damage. Using fluorescence microscopy, antibody staining, and ELISA to detect oxidative DNA damage, it was concluded that red light irradiation does not pose a safety risk to sperm DNA. The use of red light on sperm has potential in both animal conservation and human reproduction techniques. This method can also be used in conjunction with optical trapping for viable sperm selection.

The RNA Splicing Response to DNA Damage.

PubMed

Shkreta, Lulzim; Chabot, Benoit

2015-10-29

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

The RNA Splicing Response to DNA Damage

PubMed Central

Shkreta, Lulzim; Chabot, Benoit

2015-01-01

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells

PubMed Central

Burke, Russell T.; Marcus, Joshua M.; Orth, James D.

2017-01-01

Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers. PMID:28467801

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Assessment of DNA damage in a group of professional dancers during a 10-month dancing season.

PubMed

Esteves, Filipa; Teixeira, Eduardo; Amorim, Tânia; Costa, Carla; Pereira, Cristiana; Fraga, Sónia; De Andrade, Vanessa Moraes; Teixeira, João Paulo; Costa, Solange

2017-01-01

Despite the numerous health benefits of physical activity, some studies reported that increased intensity and duration may induce oxidative stress in several cellular components including DNA. The aim of this study was to assess the level of basal DNA damage as well as oxidative DNA damage in a group of professional dancers before and after a 10-month dancing season. A group of individuals from general population was also assessed as a control. The alkaline version of the comet assay was the method selected to measure both basal DNA damage and oxidative stress, since this method quantifies both endpoints. In order to measure oxidative stress, the comet assay was coupled with a lesion-specific endonuclease (formamidopyrimidine glycosylase) to detect oxidized purines. The levels of oxidative DNA damage in dancers were significantly increased after the dancing season. Pre-season levels of oxidative DNA damage were lower in dancers than those obtained from the general population, suggesting an adaptation of antioxidant system in dancers. Results of the present biomonitoring study indicate the need for more effective measures to protect ballet dancers from potentially occupational health risks related to regular intensive physical exercise.

DNA damage in children and adolescents with cardiovascular disease risk factors.

PubMed

Kliemann, Mariele; Prá, Daniel; Müller, Luiza L; Hermes, Liziane; Horta, Jorge A; Reckziegel, Miriam B; Burgos, Miria S; Maluf, Sharbel W; Franke, Silvia I R; Silva, Juliana da

2012-09-01

The risk of developing cardiovascular disease (CVD) is related to lifestyle (e.g. diet, physical activity and smoking) as well as to genetic factors. This study aimed at evaluating the association between CVD risk factors and DNA damage levels in children and adolescents. Anthropometry, diet and serum CVD risk factors were evaluated by standard procedures. DNA damage levels were accessed by the comet assay (Single cell gel electrophoresis; SCGE) and cytokinesis-blocked micronucleus (CBMN) assays in leukocytes. A total of 34 children and adolescents selected from a population sample were divided into three groups according to their level of CVD risk. Moderate and high CVD risk subjects showed significantly higher body fat and serum CVD risk markers than low risk subjects (P<0.05). High risk subjects also showed a significant increase in DNA damage, which was higher than that provided by low and moderate risk subjects according to SCGE, but not according to the CBMN assay. Vitamin C intake was inversely correlated with DNA damage by SCGE, and micronucleus (MN) was inversely correlated with folate intake. The present results indicate an increase in DNA damage that may be a consequence of oxidative stress in young individuals with risk factors for CVD, indicating that the DNA damage level can aid in evaluating the risk of CVD.

Evaluation of DNA-damaging potential of bisphenol A and its selected analogs in human peripheral blood mononuclear cells (in vitro study).

PubMed

Mokra, Katarzyna; Kuźmińska-Surowaniec, Agnieszka; Woźniak, Katarzyna; Michałowicz, Jaromir

2017-02-01

In the present study, we have investigated DNA-damaging potential of BPA and its analogs, i.e. bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF) in human peripheral blood mononuclear cells (PBMCs) using the alkaline and neutral versions of the comet assay, which allowed to evaluate DNA single strand-breaks (SSBs) and double strand-breaks (DSBs). The use of the alkaline version of comet assay made also possible to analyze the kinetics of DNA repair in PBMCs after exposure of the cells to BPA or its analogs. We have observed an increase in DNA damage in PBMCs treated with BPA or its analogs in the concentrations ranging from 0.01 to 10 μg/ml after 1 and 4 h incubation. It was noted that bisphenols studied caused DNA damage mainly via SSBs, while DNA fragmentation via double DSBs was low. The strongest changes in DNA damage were provoked by BPA and particularly BPAF, which were capable of inducing SSBs even at 0.01 μg/ml, while BPS caused the lowest changes (only at 10 μg/ml). We have also observed that PBMCs significantly repaired bisphenols-induced DNA damage but they were unable (excluding cells treated with BPS) to repair totally DNA breaks. Copyright © 2016 Elsevier Ltd. All rights reserved.

Corrupting the DNA damage response: a critical role for Rad52 in tumor cell survival.

PubMed

Lieberman, Rachel; You, Ming

2017-07-15

The DNA damage response enables cells to survive, maintain genome integrity, and to safeguard the transmission of high-fidelity genetic information. Upon sensing DNA damage, cells respond by activating this multi-faceted DNA damage response leading to restoration of the cell, senescence, programmed cell death, or genomic instability if the cell survives without proper repair. However, unlike normal cells, cancer cells maintain a marked level of genomic instability. Because of this enhanced propensity to accumulate DNA damage, tumor cells rely on homologous recombination repair as a means of protection from the lethal effect of both spontaneous and therapy-induced double-strand breaks (DSBs) in DNA. Thus, modulation of DNA repair pathways have important consequences for genomic instability within tumor cell biology and viability maintenance under high genotoxic stress. Efforts are underway to manipulate specific components of the DNA damage response in order to selectively induce tumor cell death by augmenting genomic instability past a viable threshold. New evidence suggests that RAD52, a component of the homologous recombination pathway, is important for the maintenance of tumor genome integrity. This review highlights recent reports indicating that reducing homologous recombination through inhibition of RAD52 may represent an important focus for cancer therapy and the specific efforts that are already demonstrating potential.

Push back to respond better: regulatory inhibition of the DNA double-strand break response.

PubMed

Panier, Stephanie; Durocher, Daniel

2013-10-01

Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

In vitro antioxidation activity and genoprotective effect of selected Chinese medicinal herbs.

PubMed

Szeto, Yim Tong; Wong, Shirley Ching Yee; Wong, Julia Wai Ming; Kalle, Wouter; Pak, Sok Cheon

2011-01-01

Some traditional Chinese medicinal seeds and fruits are well known for their antioxidant properties. This research aims to investigate whether Fructus Lycii, Fructus Schisandrae Chinensis, Fructus Ligustri Lucidi and Semen Cuscutae protect DNA from oxidant challenge by hydrogen peroxide (H(2)O(2)). The standard comet assay was used to assess the genoprotective effect of these medicinal herbs. Blood was taken from three healthy adults, aged from 36 to 42. Lymphocytes were isolated and treated with different concentrations of aqueous herbal extracts, while controls were treated with phosphate buffered saline. The lymphocytes were stressed with 50 μM H(2)O(2). Treated cells were embedded in agarose and layered on slides. These sandwiched lymphocytes were lysed and afterwards subjected to an electric field in an alkaline environment. Damaged DNA was pulled out from the nucleus towards the positive electrode as a comet tail; its density was related to the degree of DNA damage. Finally, the slides were stained with fluorescence dye and tails were visually scored for 100 cells. The experiment was repeated three times and DNA damage in treated cells was compared to the controls. There was no statistical difference in DNA damage among the herb treated cells and untreated cells in the comet assay. Our data demonstrated that the selected medicinal herbs did not show in vitro DNA protection in the comet assay against oxidant challenge.

Selective antitumor activity of roscovitine in head and neck cancer

PubMed Central

Biktasova, Asel; Bellinger, Gary; Yarbrough, Wendell G.; Issaeva, Natalia

2016-01-01

Radiation and chemotherapy that are commonly used to treat human cancers damage cellular DNA. DNA damage appears to be more toxic to cancer cells than normal cells, most likely due to deregulated checkpoint activation and/or deficiency in DNA repair pathways that are characteristics of many tumors. However, unwanted side effects arise as a result of DNA damage to normal cells during the treatment. Here, we show that roscovitine, a cyclin-dependent kinase (CDK) inhibitor that inhibits CDK-1, CDK-2, CDK-5, CDK-7, and CDK-9 due to competitive binding to the ATP site on the kinases, causes significant DNA damage followed by p53-dependent cell death in human papilloma virus (HPV)-positive, but not in HPV-negative, head and neck cancer cells. Since HPV positivity was a molecular marker for increased sensitivity of cells to roscovitine, we reasoned that systemic roscovitine administration would not be toxic to healthy HPV-negative tissue. Indeed, low roscovitine doses significantly inhibited the growth of HPV-associated xenografted tumors in mice without causing any detectable side effects. Given that inhibition of CDKs has been shown to inhibit replication of several viruses, we suggest that roscovitine treatment may represent a selective and safe targeted therapeutic option against HPV-positive head and neck cancer. PMID:27233076

Micro-electrophoresis: a noninvasive method of sperm selection based on membrane charge.

PubMed

Simon, Luke; Murphy, Kristin; Aston, Kenneth I; Emery, Benjamin R; Hotaling, James M; Carrell, Douglas T

2015-02-01

To develop a technique with the potential of isolating genetically fit sperm for assisted reproductive technology (ART) treatment without compromising its structural or functional competence. Observational study. University hospital. Fifty patients undergoing infertility diagnosis and 88 couples undergoing ART treatment. None. Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate before and after density gradient centrifugation (DGC), and evaluated for sperm DNA damage, histone retention, and couples' ART outcomes. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an intracytoplasmic sperm injection needle and directly analyzed for DNA damage. There was a reduction in the NCS population (95.10% ± 0.94% vs. 54.48% ± 2.39%) and an increase in the PCS population (4.28% ± 0.58% vs. 42.52% ± 2.36%) after DGC. The DNA damage was inversely proportional to %NCS (r(2) = -0.242) and directly proportional to the %PCS (r(2) = 0.206). When sperm were picked according to their charge and directly analyzed, sperm DNA damage was lower in the NCS population (3.9% ± 1.5%) compared with control (17.3% ± 3.2%) and %PCS populations (27.8% ± 6.0%). The %NCS was positively associated with fertilization rate (r(2) = 0.469) and blastocyst development (r(2) = 0.308) and inversely associated with embryo arrest (r(2) = -0.253). Implantation rate and clinical pregnancies were higher in patient groups with increased NCS. Selection of NCS through micro-electrophoresis has the potential to isolate sperm relatively free of DNA damage to be used in ART. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Genotoxic effects of deoxynivalenol in broiler chickens fed low-protein feeds.

PubMed

Awad, W A; Ghareeb, K; Dadak, A; Gille, L; Staniek, K; Hess, M; Böhm, J

2012-03-01

Deoxynivalenol (DON) is one of the most abundant and important trichothecenes in food and feed, and it is a significant contaminant due to its frequent occurrence at toxicologically relevant concentrations worldwide. Deoxynivalenol has negative influences on the health and performance of chicks. However, there is little information available regarding the effect of DON on DNA fragmentation in blood lymphocytes. In addition, the effects of Mycofix select (Biomin GmbH, Herzogenburg, Austria) supplementation to DON-contaminated broiler diets on lymphocyte DNA have not yet been demonstrated. Therefore, the aim of the present study was to establish the effect of DON on lipid peroxidation and lymphocyte DNA fragmentation in broilers and to evaluate the potential of Mycofix select in the prevention of toxin-mediated changes. Thirty-two 1-d-old (Ross 308 male) broiler chicks were randomly divided into 4 groups. The control group was fed a noncontaminated diet, and a second group was fed the same diet but supplemented with Mycofix select (0.25%). A third group of broilers was fed a diet artificially contaminated with 10 mg of feed-grade DON/kg of diet, and a fourth group was fed a DON-contaminated diet supplemented with Mycofix select. At the end of the feeding trial, blood was collected and the degree of lymphocyte DNA damage was measured in the plasma by comet assay. Deoxynivalenol increased (P = 0.016) the amount of DNA damage in chicken lymphocytes by 46.8%. Mycofix select protected lymphocyte DNA from the DON effects. To our knowledge, these are the first data on genotoxic effects of a moderate dose of DON on chicken lymphocytes. However, the thiobarbituric acid reactive substances level in liver and liver enzyme activity did not differ among the groups. In conclusion, the present study demonstrated that the diets contaminated with the mycotoxin DON at moderate levels in combination with low-protein feed are able to induce lymphocyte DNA damage in chickens. Supplementation with Mycofix select protected lymphocyte DNA and it was beneficial for maintaining the lymphocyte DNA integrity.

PARP inhibitors--current status and the walk towards early breast cancer.

PubMed

Glendenning, Jennifer; Tutt, Andrew

2011-10-01

Epithelial carcinomas in general arise as a result of the acquisition of and selection for multiple mutations in a parental somatic cell clone within the tissues of the primary organ of origin. In the last two decades genome caretakers, which function in key areas of DNA damage response, have been recognized as important tumour suppressor genes. Inactivating mutations in these genes occur both as germline and/or somatic mutations with increasing evidence of epigenetic silencing as an additional cause of loss of function. In any event, loss of function in a tumour cell pre-cursor clone leads to accelerated mutation acquisition and underpins the aetiology of the tumour. With increasing understanding of the complex network that is the DNA damage response, signaling pathways already recognized to be central to the establishment of the cancer phenotype are gaining additional roles as controllers of DNA repair. This has relevance to identification of wider populations of patients with tumours susceptible to approaches that target DNA repair deficiency. These have classically been with DNA damaging chemotherapy but the recently developed small molecule inhibitors of DNA repair enzymes such as Poly-ADP polymerases PARP-1 and PARP-2 have been shown to target tumour deficiencies in DNA repair as well sensitizing to DNA damaging therapeutics such as radiation and chemotherapy. Early phase trials with efficacy endpoints have been presented for the PARP inhibitors AG014699, olaparib, veliparib, iniparib and MK4827. The results of the first phase II trials exploring monotherapy PARP inhibitor strategies, which are based on revisiting the concept of synthetic lethality, have emerged and are reviewed herein. The clinical trials that have or are exploring combinations with DNA damaging therapy in these contexts are discussed with particular reference to breast cancer, as are biomarkers that have been proposed and are being investigated to develop optimal drug schedule and patient selection criteria for these DNA repair targeting approaches. Copyright © 2011 Elsevier Ltd. All rights reserved.

Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells.

PubMed

Chang, Hsueh-Wei; Li, Ruei-Nian; Wang, Hui-Ru; Liu, Jing-Ru; Tang, Jen-Yang; Huang, Hurng-Wern; Chan, Yu-Hsuan; Yen, Ching-Yu

2017-01-01

Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N -acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells.

Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells

PubMed Central

Chang, Hsueh-Wei; Li, Ruei-Nian; Wang, Hui-Ru; Liu, Jing-Ru; Tang, Jen-Yang; Huang, Hurng-Wern; Chan, Yu-Hsuan; Yen, Ching-Yu

2017-01-01

Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells. PMID:28936177

Repair of DNA-polypeptide crosslinks by human excision nuclease

NASA Astrophysics Data System (ADS)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

Consequences of acute oxidative stress in Leishmania amazonensis: From telomere shortening to the selection of the fittest parasites.

PubMed

da Silva, Marcelo Santos; Segatto, Marcela; Pavani, Raphael Souza; Gutierrez-Rodrigues, Fernanda; Bispo, Vanderson da Silva; de Medeiros, Marisa Helena Gennari; Calado, Rodrigo Tocantins; Elias, Maria Carolina; Cano, Maria Isabel Nogueira

2017-01-01

Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects millions of people around the world. During infection, the parasites use different strategies to survive the host's defenses, including overcoming exposure to reactive oxidant species (ROS), responsible for causing damage to lipids, proteins and DNA. This damage especially affects telomeres, which frequently results in genome instability, senescence and cell death. Telomeres are the physical ends of the chromosomes composed of repetitive DNA coupled with proteins, whose function is to protect the chromosomes termini and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (H 2 O 2 ) for 1h, which was able to increase intracellular ROS levels. In addition, oxidative stress induced DNA damage, as confirmed by 8-oxodGuo quantification and TUNEL assays and the dissociation of LaRPA-1 from the 3' G-overhang, leading to telomere shortening. Moreover, LaRPA-1 was observed to interact with newly formed C-rich single-stranded telomeric DNA, probably as a consequence of the DNA damage response. Nonetheless, acute oxidative stress caused the death of some of the L. amazonensis population and induced cell cycle arrest at the G2/M phase in survivor parasites, which were able to continue proliferating and replicating DNA and became more resistant to oxidative stress. Taken together, these results suggest that adaptation occurs through the selection of the fittest parasites in terms of repairing oxidative DNA damage at telomeres and maintaining genome stability in a stressful environment. Copyright © 2016 Elsevier B.V. All rights reserved.

The possible DNA damage induced by environmental organic compounds: The case of Nonylphenol.

PubMed

Noorimotlagh, Zahra; Mirzaee, Seyyed Abbas; Ahmadi, Mehdi; Jaafarzadeh, Neemat; Rahim, Fakher

2018-08-30

Human impact on the environment leads to the release of many pollutants that produce artificial compounds, which can have harmful effects on the body's endocrine system; these are known as endocrine disruptors (EDs). Nonylphenol (NP) is a chemical compound with a nonyl group that is attached to a phenol ring. NP-induced H 2 AX is a sensitive genotoxic biomarker for detecting possible DNA damage; it also causes male infertility and carcinogenesis. We attempt to comprehensively review all the available evidence about the different ways with descriptive mechanisms for explaining the possible DNA damage that is induced by NP. We systematically searched several databases, including PubMed, Scopus, Web of Science, and gray literature, such as Google Scholar by using medical subheading (MeSH) terms and various combinations of selected keywords from January 1970 to August 2017. The initial search identified 62,737 potentially eligible studies; of these studies, 33 were included according to the established inclusion criteria. Thirty-three selected studies, include the topics of animal model (n = 21), cell line (n = 6), human model (n = 4), microorganisms (n = 1), solid DNA (n = 1), infertility (n = 4), apoptosis (n = 6), and carcinogenesis (n = 3). This review highlighted the possible deleterious effects of NP on DNA damage through the ability to produce ROS/RNS. Finally, it is significant to observe caution at this stage with the continued use of environmental pollutants such as NP, which may induce DNA damage and apoptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

PubMed Central

Calo, Eliezer; Gu, Bo; Bowen, Margot E.; Aryan, Fardin; Zalc, Antoine; Liang, Jialiang; Flynn, Ryan A.; Swigut, Tomek; Chang, Howard Y.; Attardi, Laura D.; Wysocka, Joanna

2018-01-01

Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis1,2. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis3,4, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis5, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations. PMID:29364875

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders.

PubMed

Calo, Eliezer; Gu, Bo; Bowen, Margot E; Aryan, Fardin; Zalc, Antoine; Liang, Jialiang; Flynn, Ryan A; Swigut, Tomek; Chang, Howard Y; Attardi, Laura D; Wysocka, Joanna

2018-02-01

Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

PubMed

Paini, Alicia; Scholz, Gabriele; Marin-Kuan, Maricel; Schilter, Benoît; O'Brien, John; van Bladeren, Peter J; Rietjens, Ivonne M C M

2011-09-01

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells.

PubMed

Hollenbach, Jessica P; Resch, Alissa M; Palakodeti, Dasaradhi; Graveley, Brenton R; Heinen, Christopher D

2011-01-01

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.

Loss of DNA Mismatch Repair Imparts a Selective Advantage in Planarian Adult Stem Cells

PubMed Central

Hollenbach, Jessica P.; Resch, Alissa M.; Palakodeti, Dasaradhi; Graveley, Brenton R.; Heinen, Christopher D.

2011-01-01

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis. PMID:21747960

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Rongxin; Mullins, Elwood A.; Shen, Xing‐Xing

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct frommore » that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.« less

Linking loss of sodium-iodide symporter expression to DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyckesvärd, Madeleine Nordén; Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg; Kapoor, Nirmal

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1 nM) calicheamicin blocked NIS mRNA expression andmore » transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress. - Highlights: • DNA damage inhibits polarized iodide transport in normal thyroid cells. • Down-regulation of NIS expression is mediated by activation of the ATM kinase. • Long-term ATM inhibition also represses NIS-mediated iodide transport. • IGF-1 rescues NIS expression and iodide transport in DNA-damaged cells.« less

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

PubMed

Alvarado-Cruz, Isabel; Sánchez-Guerra, Marco; Hernández-Cadena, Leticia; De Vizcaya-Ruiz, Andrea; Mugica, Violeta; Pelallo-Martínez, Nadia Azenet; Solís-Heredia, María de Jesús; Byun, Hyang-Min; Baccarelli, Andrea; Quintanilla-Vega, Betzabet

2017-01-01

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM 10 (≤10μm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM 10 exposure showed that PM 10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m 3 in benzo[b]fluoranthene content of PM 10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM 10 -associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.

PubMed

Li, Cuiping; Wang, Hailin

2015-08-07

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1×10(-19)mol in mass and 2.9pM in concentration. Copyright © 2015 Elsevier B.V. All rights reserved.

Study of terahertz-radiation-induced DNA damage in human blood leukocytes

NASA Astrophysics Data System (ADS)

Angeluts, A. A.; Gapeyev, A. B.; Esaulkov, M. N.; Kosareva, O. G.; Matyunin, S. N.; Nazarov, M. M.; Pashovkin, T. N.; Solyankin, P. M.; Cherkasova, O. P.; Shkurinov, A. P.

2014-03-01

We have carried out the studies aimed at assessing the effect of terahertz radiation on DNA molecules in human blood leukocytes. Genotoxic testing of terahertz radiation was performed in three different oscillation regimes, the blood leukocytes from healthy donors being irradiated for 20 minutes with the mean intensity of 8 - 200 μW cm-2 within the frequency range of 0.1 - 6.5 THz. Using the comet assay it is shown that in the selected regimes such radiation does not induce a direct DNA damage in viable human blood leukocytes.

Identification of ATR-Chk1 pathway inhibitors that selectively target p53-deficient cells without directly suppressing ATR catalytic activity

PubMed Central

Kawasumi, Masaoki; Bradner, James E.; Tolliday, Nicola; Thibodeau, Renee; Sloan, Heather; Brummond, Kay M.; Nghiem, Paul

2014-01-01

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells where the G1/S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2/M checkpoints mediated by the ATR and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. PMID:25336189

Identification of ATR-Chk1 pathway inhibitors that selectively target p53-deficient cells without directly suppressing ATR catalytic activity.

PubMed

Kawasumi, Masaoki; Bradner, James E; Tolliday, Nicola; Thibodeau, Renee; Sloan, Heather; Brummond, Kay M; Nghiem, Paul

2014-12-15

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1-S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2-M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. ©2014 American Association for Cancer Research.

Western and Chinese antirheumatic drug-induced T cell apoptotic DNA damage uses different caspase cascades and is independent of Fas/Fas ligand interaction.

PubMed

Lai, J H; Ho, L J; Lu, K C; Chang, D M; Shaio, M F; Han, S H

2001-06-01

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.

Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

NASA Astrophysics Data System (ADS)

Lu, Tao; Wu, Honglu; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Wong, Michael

2016-07-01

Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect initial transcriptional responses to bleomycin treatment in the selected genes in the DNA damage signaling pathways.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

PubMed

Zhang, Xiaohong; Jeffs, Graham; Ren, Xiaolin; O'Donovan, Peter; Montaner, Beatriz; Perrett, Conal M; Karran, Peter; Xu, Yao-Zhong

2007-03-01

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

PubMed

Zegura, B; Gajski, G; Straser, A; Garaj-Vrhovac, V; Filipič, M

2011-12-24

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

Genes and Junk in Plant Mitochondria—Repair Mechanisms and Selection

PubMed Central

Christensen, Alan C.

2014-01-01

Plant mitochondrial genomes have very low mutation rates. In contrast, they also rearrange and expand frequently. This is easily understood if DNA repair in genes is accomplished by accurate mechanisms, whereas less accurate mechanisms including nonhomologous end joining or break-induced replication are used in nongenes. An important question is how different mechanisms of repair predominate in coding and noncoding DNA, although one possible mechanism is transcription-coupled repair (TCR). This work tests the predictions of TCR and finds no support for it. Examination of the mutation spectra and rates in genes and junk reveals what DNA repair mechanisms are available to plant mitochondria, and what selective forces act on the repair products. A model is proposed that mismatches and other DNA damages are repaired by converting them into double-strand breaks (DSBs). These can then be repaired by any of the DSB repair mechanisms, both accurate and inaccurate. Natural selection will eliminate coding regions repaired by inaccurate mechanisms, accounting for the low mutation rates in genes, whereas mutations, rearrangements, and expansions generated by inaccurate repair in noncoding regions will persist. Support for this model includes the structure of the mitochondrial mutS homolog in plants, which is fused to a double-strand endonuclease. The model proposes that plant mitochondria do not distinguish a damaged or mismatched DNA strand from the undamaged strand, they simply cut both strands and perform homology-based DSB repair. This plant-specific strategy for protecting future generations from mitochondrial DNA damage has the side effect of genome expansions and rearrangements. PMID:24904012

DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells.

PubMed

Lim, Hui Kheng; Gurung, Resham Lal; Hande, M Prakash

2017-12-01

Silver nanoparticles (Ag-np) were reported to be toxic to eukaryotic cells. These potentially detrimental effects of Ag-np can be advantageous in experimental therapeutics. They are currently being employed to enhance the therapeutic efficacy of cancer drugs. In this study, we demonstrate that Ag-np treatment trigger the activation of DNA-PKcs and JNK pathway at selected doses, presumably as a physiologic response to DNA damage and repair in normal and malignant cells. Ag-np altered the telomere dynamics by disrupting the shelterin complex located at the telomeres and telomere lengths. The genotoxic effect of Ag-np was not restricted to telomeres but the entire genome as Ag-np induced γ-H2AX foci formation, an indicator of global DNA damage. Inhibition of DNA-PKcs activity sensitised the cancer cells towards the cytotoxicity of Ag-np and substantiated the damaging effect of Ag-np at telomeres in human cancer cells. Abrogation of JNK mediated DNA repair and extensive damage of telomeres led to greater cell death following Ag-np treatment in DNA-PKcs inhibited cancer cells. Collectively, this study suggests that improved anti-proliferative and cytotoxic effects of Ag-np treatment in cancer cells can be achieved by the inhibition of DNA-PKcs. Copyright © 2017 Elsevier B.V. All rights reserved.

Approaches for characterizing threshold dose-response relationships for DNA-damage pathways involved in carcinogenicity in vivo and micronuclei formation in vitro.

PubMed

Clewell, Rebecca A; Andersen, Melvin E

2016-05-01

Assessing the shape of dose-response curves for DNA-damage in cellular systems and for the consequences of DNA damage in intact animals remains a controversial topic. This overview looks at aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of cellular DNA-damage/repair and their role in defining the shape of dose-response curves using an in vivo example with formaldehyde and in vitro examples for micronuclei (MN) formation with several test compounds. Formaldehyde is both strongly mutagenic and an endogenous metabolite in cells. With increasing inhaled concentrations, there were transitions in gene changes, from activation of selective stress pathway genes at low concentrations, to activation of pathways for cell-cycle control, p53-DNA damage, and stem cell niche pathways at higher exposures. These gene expression changes were more consistent with dose-dependent transitions in the PD responses to formaldehyde in epithelial cells in the intact rat rather than the low-dose linear extrapolation methods currently used for carcinogens. However, more complete PD explanations of non-linear dose response for creation of fixed damage in cells require detailed examination of cellular responses in vitro using measures of DNA damage and repair that are not easily accessible in the intact animal. In the second section of the article, we illustrate an approach from our laboratory that develops fit-for-purpose, in vitro assays and evaluates the PD of DNA damage and repair through studies using prototypical DNA-damaging agents. Examination of a broad range of responses in these cells showed that transcriptional upregulation of cell cycle control and DNA repair pathways only occurred at doses higher than those causing overt damage fixed damage-measured as MN formation. Lower levels of damage appear to be handled by post-translational repair process using pre-existing proteins. In depth evaluation of the PD properties of one such post-translational process (formation of DNA repair centers; DRCs) has indicated that the formation of DRCs and their ability to complete repair before replication are consistent with threshold behaviours for mutagenesis and, by extension, with chemical carcinogenesis. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

PubMed Central

Lountos, George T; Tropea, Joseph E; Zhang, Di; Jobson, Andrew G; Pommier, Yves; Shoemaker, Robert H; Waugh, David S

2009-01-01

Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity. PMID:19177354

New orally active DNA minor groove binding small molecule CT-1 acts against breast cancer by targeting tumor DNA damage leading to p53-dependent apoptosis.

PubMed

Saini, Karan Singh; Hamidullah; Ashraf, Raghib; Mandalapu, Dhanaraju; Das, Sharmistha; Siddiqui, Mohd Quadir; Dwivedi, Sonam; Sarkar, Jayanta; Sharma, Vishnu Lal; Konwar, Rituraj

2017-04-01

Targeting tumor DNA damage and p53 pathway is a clinically established strategy in the development of cancer chemotherapeutics. Majority of anti-cancer drugs are delivered through parenteral route for reasons like severe toxicity, lack of stability, and poor enteral absorption. Current DNA targeting drugs in clinical like anthracycline suffers from major drawbacks like cardiotoxicity. Here, we report identification of a new orally active small molecule curcumin-triazole conjugate (CT-1) with significant anti-breast cancer activity in vitro and in vivo. CT-1 selectively and significantly inhibits viability of breast cancer cell lines; retards cells cycle progression at S phase and induce mitochondrial-mediated cell apoptosis. CT-1 selectively binds to minor groove of DNA and induces DNA damage leading to increase in p53 along with decrease in its ubiquitination. Inhibition of p53 with pharmacological inhibitor as well as siRNA revealed the necessity of p53 in CT-1-mediated anti-cancer effects in breast cancer cells. Studies using several other intact p53 and deficient p53 cancer cell lines further confirmed necessity of p53 in CT-1-mediated anti-cancer response. Pharmacological inhibition of pan-caspase showed CT-1 induces caspase-dependent cell death in breast cancer cells. Most interestingly, oral administration of CT-1 induces significant inhibition of tumor growth in LA-7 syngeneic orthotropic rat mammary tumor model. CT-1 treated mammary tumor shows enhancement in DNA damage, p53 upregulation, and apoptosis. Collectively, CT-1 exhibits potent anti-cancer effect both in vitro and in vivo and could serve as a safe orally active lead for anti-cancer drug development. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Ancestor of land plants acquired the DNA-3-methyladenine glycosylase (MAG) gene from bacteria through horizontal gene transfer.

PubMed

Fang, Huimin; Huangfu, Liexiang; Chen, Rujia; Li, Pengcheng; Xu, Shuhui; Zhang, Enying; Cao, Wei; Liu, Li; Yao, Youli; Liang, Guohua; Xu, Chenwu; Zhou, Yong; Yang, Zefeng

2017-08-24

The origin and evolution of land plants was an important event in the history of life and initiated the establishment of modern terrestrial ecosystems. From water to terrestrial environments, plants needed to overcome the enhanced ultraviolet (UV) radiation and many other DNA-damaging agents. Evolving new genes with the function of DNA repair is critical for the origin and radiation of land plants. In bacteria, the DNA-3-methyladenine glycosylase (MAG) recognizes of a variety of base lesions and initiates the process of the base excision repair for damaged DNA. The homologs of MAG gene are present in all major lineages of streptophytes, and both the phylogenic and sequence similarity analyses revealed that green plant MAG gene originated through an ancient horizontal gene transfer (HGT) event from bacteria. Experimental evidence demonstrated that the expression of the maize ZmMAG gene was induced by UV and zeocin, both of which are known as DNA-damaging agents. Further investigation revealed that Streptophyta MAG genes had undergone positive selection during the initial evolutionary period in the ancestor of land plants. Our findings demonstrated that the ancient HGT of MAG to the ancestor of land plants probably played an important role in preadaptation to DNA-damaging agents in terrestrial environments.

DNA damage response in monozygotic twins discordant for smoking habits.

PubMed

Marcon, Francesca; Carotti, Daniela; Andreoli, Cristina; Siniscalchi, Ester; Leopardi, Paola; Caiola, Stefania; Biffoni, Mauro; Zijno, Andrea; Medda, Emanuela; Nisticò, Lorenza; Rossi, Sabrina; Crebelli, Riccardo

2013-03-01

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Unexpected effect of dry olive leaf extract on the level of DNA damage in lymphocytes of lead intoxicated workers, before and after CaNa2EDTA chelation therapy.

PubMed

Čabarkapa, Andrea; Dekanski, Dragana; Živković, Lada; Milanović-Čabarkapa, Mirjana; Bajić, Vladan; Topalović, Dijana; Giampieri, Francesca; Gasparrini, Massimiliano; Battino, Maurizio; Spremo-Potparević, Biljana

2017-08-01

The CaNa 2 EDTA chelation therapy is often practiced with antioxidant supplementation. Dry olive leaf extract (DOLE) is natural product with antioxidant and DNA protective properties. The effects of DOLE on the levels of DNA damage were investigated ex vivo in peripheral blood lymphocytes (PBLs) of 19 workers occupationally exposed to lead (Pb), before and after CaNa 2 EDTA chelation therapy. DOLE demonstrated pronounced radical scavenging activity in concentrations ≥1 mg/mL, and showed no genotoxicity per se, in concentrations 0.125-1 mg/mL. The level of DNA damage in PBLs of workers before chelation therapy was elevated (24.21 ± 14.26) compared to controls (6.0 ± 3.37). The incubation of PBLs before chelation therapy with selected concentration of DOLE lead to a severe increase of DNA damage (64.03 ± 20.96), exhibiting prooxidant rather than antioxidant effect. After the five-day CaNa 2 EDTA chelation regimen, DNA damage in PBLs of workers decreased (8.26 ± 4.62) significantly compared to baseline. Treatment of PBLs with DOLE after chelation, again produced high level of damage (41.82 ± 23.17) and the acute prooxidant effects of DOLE remained, but, DNA damage was less severe than before chelation. The DOLE exhibits prooxidant effect in presence of Pb in lymphocytes of exposed workers, and its effect is less pronounced following the removal of Pb after standard chelation therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Junqiang; Doi, Hiroshi; Saar, Matthias

2013-12-01

Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome.more » The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.« less

Diet-Induced Weight Loss Reduces DNA Damage and Cardiometabolic Risk Factors in Overweight/Obese Women with Polycystic Ovary Syndrome.

PubMed

Soares, Nayara Pereira; Santos, Ana Celly Souza dos; Costa, Eduardo Caldas; Azevedo, George Dantas; Damasceno, Débora Cristina; Fayh, Ana Paula Trussardi; Lemos, Telma Maria Araújo Moura

2016-01-01

We aimed to investigate the impact of following a diet to induce weight loss (500 kcal deficit per day) over DNA damage and cardiometabolic risk factors in women with overweight/obesity diagnosed with polycystic ovary syndrome (PCOS). A study was conducted in Natal, RN, Brazil selecting overweight/obese (body mass index ≥25 and <39 kg/m2) women (18-35 years). The levels of DNA damage were assessed by a single cell gel electrophoresis. Repeated 24 h dietary recall questionnaires, anthropometry, biochemical profile and sex hormones were collected at baseline and after 12 weeks of intervention. Women exhibiting a decrease in the markers of DNA damage: tail intensity (24.35 ± 5.86 - pre diet vs. 17.15 ± 5.04 - post-diet; p < 0.001) and tail moment (20.47 ± 7.85 - pre diet vs. 14.13 ± 6.29 - post-diet; p < 0.002). Reduction of calorie intake, weight loss, decreased sexual hormone and cardiometabolic markers such as insulin, homeostasis model assessment of insulin resistance and low-density lipoprotein cholesterol were verified In the multivariate regression analysis, quantitative insulin sensitivity check index and progesterone were responsible for the variation markers in DNA damage before the diet, losing its influence upon diet. DNA damage and the impact of cardiometabolic risk factors decreased after the intervention in women with PCOS, indicating the relevance of a nutritional approach in this group of patients. © 2016 S. Karger AG, Basel.

TATA Binding Protein Discriminates between Different Lesions on DNA, Resulting in a Transcription Decrease

PubMed Central

Coin, Frédéric; Frit, Philippe; Viollet, Benoit; Salles, Bernard; Egly, Jean-Marc

1998-01-01

DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position −29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage. PMID:9632775

Diseases Associated with Defective Responses to DNA Damage

PubMed Central

O’Driscoll, Mark

2012-01-01

Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways. PMID:23209155

The effect of 2-[(aminopropyl)amino] ethanethiol on fission-neutron-induced DNA damage and repair.

PubMed Central

Grdina, D. J.; Sigdestad, C. P.; Dale, P. J.; Perrin, J. M.

1989-01-01

The effect(s) of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) on fission-neutron-induced DNA damage and repair in V79 Chinese hamster cells was determined by using a neutral filter elution procedure (pH 7.2). When required, WR1065, at a final working concentration of 4 mM, was added to the culture medium, either 30 min before and during irradiation with fission spectrum neutrons (beam energy of 0.85 MeV) from the JANUS research reactor, or for selected intervals of time following exposure. The frequency of neutron-induced DNA strand breaks as measured by neutral elution as a function of dose equalled that observed for 60Co gamma-ray-induced damage (relative biological effectiveness of one). In contrast to the protective effect exhibited by WR1065 in reducing 60Co-induced DNA damage, WR1065 was ineffective in reducing or protecting against induction of DNA strand breaks by JANUS neutrons. The kinetics of DNA double-strand rejoining were measured following neutron irradiation. In the absence of WR1065, considerable DNA degradation by cellular enzymes was observed. This process was inhibited when WR1065 was present. These results indicate that, under the conditions used, the quality (i.e. nature), rather than quantity, of DNA lesions (measured by neutral elution) formed by neutrons was significantly different from that formed by gamma-rays. PMID:2667608

Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970

PubMed Central

Boucher, Diane M.; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A.; Milton, Sean; Murphy, Cheryl E.; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M.; Pollard, John R.

2014-01-01

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients. PMID:25010037

Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

PubMed

Hall, Amy B; Newsome, Dave; Wang, Yuxin; Boucher, Diane M; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A; Milton, Sean; Murphy, Cheryl E; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M; Pollard, John R

2014-07-30

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

Electron Nuclear Dynamics Simulations of Proton Cancer Therapy Reactions: Water Radiolysis and Proton- and Electron-Induced DNA Damage in Computational Prototypes.

PubMed

Teixeira, Erico S; Uppulury, Karthik; Privett, Austin J; Stopera, Christopher; McLaurin, Patrick M; Morales, Jorge A

2018-05-06

Proton cancer therapy (PCT) utilizes high-energy proton projectiles to obliterate cancerous tumors with low damage to healthy tissues and without the side effects of X-ray therapy. The healing action of the protons results from their damage on cancerous cell DNA. Despite established clinical use, the chemical mechanisms of PCT reactions at the molecular level remain elusive. This situation prevents a rational design of PCT that can maximize its therapeutic power and minimize its side effects. The incomplete characterization of PCT reactions is partially due to the health risks associated with experimental/clinical techniques applied to human subjects. To overcome this situation, we are conducting time-dependent and non-adiabatic computer simulations of PCT reactions with the electron nuclear dynamics (END) method. Herein, we present a review of our previous and new END research on three fundamental types of PCT reactions: water radiolysis reactions, proton-induced DNA damage and electron-induced DNA damage. These studies are performed on the computational prototypes: proton + H₂O clusters, proton + DNA/RNA bases and + cytosine nucleotide, and electron + cytosine nucleotide + H₂O. These simulations provide chemical mechanisms and dynamical properties of the selected PCT reactions in comparison with available experimental and alternative computational results.

The Quinone Based Antitumor Agent Sepantronium Bromide (YM155) Causes Oxygen Independent Redox Activated Oxidative DNA Damage.

PubMed

Wani, Tasaduq Hussain; Surendran, Sreeraj; Jana, Anal; Chakrabarty, Anindita; Chowdhury, Goutam

2018-06-13

Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155's primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays we showed that YM155 cause extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions indicating that hydrogen peroxide and oxygen is not necessary. Although YM155 is a quinone, it does not follow a typical quinone mechanism. Consistent with these observations a mechanism has been proposed that suggests that YM155 can cause oxidative DNA cleavage upon two electron reductive activation.

Miscoding and mutagenic properties of 8-oxoguanine and abasic sites: Ubiquitous lesions in damaged DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grollman, A.P.; Takeshita, Masaru

1995-12-31

More than twenty oxidatively-damaged bases, including 8-oxoguanine, have been found to occur in genomic DNA. Some of these lesions block DNA replication and are potentially lethal; others generate mutations which can initiate carcinogenesis and promote cellular aging. In this report, the authors focus attention on the mutagenicity and repair of 8-oxoguanine. Kasai and Nishimura`s discovery that hydroxyl radicals react with guanine residues in DNA to form 8-oxoguanine and the development of sensitive methods for the detection and quantitation of this modified base led to the observation that approximately 1 in 10{sup 5} guanine residues in mammalian DNA are oxidized atmore » the C-8 position. DNA containing 8-oxoguanine and synthetic analogs of the abasic site have been used to investigate the miscoding and mutagenic potential of these ubiquitous lesions. Studies in the laboratory were facilitated by the development of solid state synthetic methods by which these lesions could be introduced at defined positions in DNA. In this paper, the authors review studies in which 8-oxoguanine and abasic sites have been used in model systems to explore various early events in the replication of selectively damaged DNA.« less

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abbasi, Rashda; Efferth, Thomas; Kuhmann, Christine

2012-03-15

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC{sub 50} values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol wasmore » the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC{sub 50} values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy options in NER-deficient tumors.« less

TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

PubMed Central

Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

2015-01-01

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

PARP Inhibitors in Reproductive System Cancers: Current Use and Developments.

PubMed

O'Sullivan Coyne, Geraldine; Chen, Alice P; Meehan, Robert; Doroshow, James H

2017-02-01

The repair of DNA damage is a critical cellular process governed by multiple biochemical pathways that are often found to be defective in cancer cells. The poly(ADP-ribose) polymerase (PARP) family of proteins controls response to single-strand DNA breaks by detecting these damaged sites and recruiting the proper factors for repair. Blocking this pathway forces cells to utilize complementary mechanisms to repair DNA damage. While PARP inhibition may not, in itself, be sufficient to cause tumor cell death, inhibition of DNA repair with PARP inhibitors is an effective cytotoxic strategy when it is used in patients who carry other defective DNA-repair mechanisms, such as mutations in the genes BRCA 1 and 2. This discovery has supported the development of PARP inhibitors (PARPi), agents that have proven effective against various types of tumors that carry BRCA mutations. With the application of next-generation sequencing of tumors, there is increased interest in looking beyond BRCA mutations to identify genetic and epigenetic aberrations that might lead to similar defects in DNA repair, conferring susceptibility to PARP inhibition. Identification of these genetic lesions and the development of screening assays for their detection may allow for the selection of patients most likely to respond to this class of anticancer agents. This article provides an overview of clinical trial results obtained with PARPi and describes the companion diagnostic assays being established for patient selection. In addition, we review known mechanisms for resistance to PARPi and potential strategies for combining these agents with other types of therapy.

Unexpected Function of the Glucanosyltransferase Gas1 in the DNA Damage Response Linked to Histone H3 Acetyltransferases in Saccharomyces cerevisiae

PubMed Central

Eustice, Moriah; Pillus, Lorraine

2014-01-01

Chromatin organization and structure are crucial for transcriptional regulation, DNA replication, and damage repair. Although initially characterized in remodeling cell wall glucans, the β-1,3-glucanosyltransferase Gas1 was recently discovered to regulate transcriptional silencing in a manner separable from its activity at the cell wall. However, the function of Gas1 in modulating chromatin remains largely unexplored. Our genetic characterization revealed that GAS1 had critical interactions with genes encoding the histone H3 lysine acetyltransferases Gcn5 and Sas3. Specifically, whereas the gas1gcn5 double mutant was synthetically lethal, deletion of both GAS1 and SAS3 restored silencing in Saccharomyces cerevisiae. The loss of GAS1 also led to broad DNA damage sensitivity with reduced Rad53 phosphorylation and defective cell cycle checkpoint activation following exposure to select genotoxins. Deletion of SAS3 in the gas1 background restored both Rad53 phosphorylation and checkpoint activation following exposure to genotoxins that trigger the DNA replication checkpoint. Our analysis thus uncovers previously unsuspected functions for both Gas1 and Sas3 in DNA damage response and cell cycle regulation. PMID:24532730

Cisplatin: mode of cytotoxic action and molecular basis of resistance.

PubMed

Siddik, Zahid H

2003-10-20

Cisplatin is one of the most potent antitumor agents known, displaying clinical activity against a wide variety of solid tumors. Its cytotoxic mode of action is mediated by its interaction with DNA to form DNA adducts, primarily intrastrand crosslink adducts, which activate several signal transduction pathways, including those involving ATR, p53, p73, and MAPK, and culminate in the activation of apoptosis. DNA damage-mediated apoptotic signals, however, can be attenuated, and the resistance that ensues is a major limitation of cisplatin-based chemotherapy. The mechanisms responsible for cisplatin resistance are several, and contribute to the multifactorial nature of the problem. Resistance mechanisms that limit the extent of DNA damage include reduced drug uptake, increased drug inactivation, and increased DNA adduct repair. Origins of these pharmacologic-based mechanisms, however, are at the molecular level. Mechanisms that inhibit propagation of the DNA damage signal to the apoptotic machinery include loss of damage recognition, overexpression of HER-2/neu, activation of the PI3-K/Akt (also known as PI3-K/PKB) pathway, loss of p53 function, overexpression of antiapoptotic bcl-2, and interference in caspase activation. The molecular signature defining the resistant phenotype varies between tumors, and the number of resistance mechanisms activated in response to selection pressures dictates the overall extent of cisplatin resistance.

Pre-neurodegeneration of mitral cells in the pcd mutant mouse is associated with DNA damage, transcriptional repression, and reorganization of nuclear speckles and Cajal bodies.

PubMed

Valero, Jorge; Berciano, Maria T; Weruaga, Eduardo; Lafarga, Miguel; Alonso, José R

2006-11-01

DNA damage and impairment of its repair underlie several neurodegenerative diseases. The Purkinje cell degeneration (pcd) mutation causes the loss of Nna1 expression and is associated with a selective and progressive degeneration of specific neuronal populations, including mitral cells in the olfactory bulb. Using an in situ transcription assay, molecular markers for both nuclear compartments and components of the DNA damage/repair pathway, and ultrastructural analysis, here we demonstrate that the pcd mutation induces the formation of DNA damage/repair foci in mitral cells. Furthermore, this effect is associated with transcriptional inhibition, heterochromatinization, nucleolar segregation and the reorganization of nuclear speckles of splicing factors and Cajal bodies. The most significant cytoplasmic alteration observed was a partial replacement of rough endoplasmic reticulum cisternae by a larger amount of free ribosomes, while other organelles were structurally preserved. The tools employed in this work may be of use for the early detection of predegenerative processes in neurodegenerative disorders and for validating rescue strategies.

Systematic random sampling of the comet assay.

PubMed

McArt, Darragh G; Wasson, Gillian R; McKerr, George; Saetzler, Kurt; Reed, Matt; Howard, C Vyvyan

2009-07-01

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

PubMed

Abbas, Hussein H K; Alhamoudi, Kheloud M H; Evans, Mark D; Jones, George D D; Foster, Steven S

2018-04-16

Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC

NASA Astrophysics Data System (ADS)

Chen, Xuejing; Velmurugu, Yogambigai; Zheng, Guanqun; Park, Beomseok; Shim, Yoonjung; Kim, Youngchang; Liu, Lili; van Houten, Bennett; He, Chuan; Ansari, Anjum; Min, Jung-Hyun

2015-01-01

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.

Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect

PubMed Central

Salvati, Erica; Leonetti, Carlo; Rizzo, Angela; Scarsella, Marco; Mottolese, Marcella; Galati, Rossella; Sperduti, Isabella; Stevens, Malcolm F.G.; D’Incalci, Maurizio; Blasco, Maria; Chiorino, Giovanna; Bauwens, Serge; Horard, Béatrice; Gilson, Eric; Stoppacciaro, Antonella; Zupi, Gabriella; Biroccio, Annamaria

2007-01-01

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors γ-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA–binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy. PMID:17932567

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage.

PubMed

Xu, Limei; Zhang, Chongmiao; Xu, Pengcheng; Wang, Xiaochang C

2018-03-01

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants - ultraviolet (UV) and chlorine - on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm 2 ). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm 2 ) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm 2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability. Copyright © 2017. Published by Elsevier B.V.

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture

PubMed Central

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. PMID:27821713

A bio-inspired structural health monitoring system based on ambient vibration

NASA Astrophysics Data System (ADS)

Lin, Tzu-Kang; Kiremidjian, Anne; Lei, Chi-Yang

2010-11-01

A structural health monitoring (SHM) system based on naïve Bayesian (NB) damage classification and DNA-like expression data was developed in this research. Adapted from the deoxyribonucleic acid (DNA) array concept in molecular biology, the proposed structural health monitoring system is constructed utilizing a double-tier regression process to extract the expression array from the structural time history recorded during external excitations. The extracted array is symbolized as the various genes of the structure from the viewpoint of molecular biology and reflects the possible damage conditions prevalent in the structure. A scaled down, six-story steel building mounted on the shaking table of the National Center for Research on Earthquake Engineering (NCREE) was used as the benchmark. The structural response at different damage levels and locations under ambient vibration was collected to support the database for the proposed SHM system. To improve the precision of detection in practical applications, the system was enhanced by an optimization process using the likelihood selection method. The obtained array representing the DNA array of the health condition of the structure was first evaluated and ranked. A total of 12 groups of expression arrays were regenerated from a combination of four damage conditions. To keep the length of the array unchanged, the best 16 coefficients from every expression array were selected to form the optimized SHM system. Test results from the ambient vibrations showed that the detection accuracy of the structural damage could be greatly enhanced by the optimized expression array, when compared to the original system. Practical verification also demonstrated that a rapid and reliable result could be given by the final system within 1 min. The proposed system implements the idea of transplanting the DNA array concept from molecular biology into the field of SHM.

Tripeptidyl peptidase II plays a role in the radiation response of selected primary cell types but not based on nuclear translocation and p53 stabilization.

PubMed

Firat, Elke; Tsurumi, Chizuko; Gaedicke, Simone; Huai, Jisen; Niedermann, Gabriele

2009-04-15

The giant cytosolic protease tripeptidyl peptidase II (TPPII) was recently proposed to play a role in the DNA damage response. Shown were nuclear translocation of TPPII after gamma-irradiation, lack of radiation-induced p53 stabilization in TPPII-siRNA-treated cells, and complete tumor regression in mice after gamma-irradiation when combined with TPPII-siRNA silencing or a protease inhibitor reported to inhibit TPPII. This suggested that TPPII could be a novel target for tumor radiosensitization and prompted us to study radiation responses using TPPII-knockout mice. Neither the sensitivity to total body irradiation nor the radiosensitivity of resting lymphoid cells, which both strongly depend on p53, was altered in the absence of TPPII. Functional integrity of p53 in TPPII-knockout cells is further shown by a proper G(1) arrest and by the accumulation of p53 and its transcriptional targets, p21, Bax, and Fas, on gamma-irradiation. Furthermore, we could not confirm radiation-induced nuclear translocation of TPPII. Nevertheless, after gamma-irradiation, we found slightly increased mitotic catastrophe of TPPII-deficient primary fibroblasts and increased apoptosis of TPPII-deficient activated CD8(+) T cells. The latter was accompanied by delayed resolution of the DNA double-strand break marker gammaH2AX. This could, however, be due to increased apoptotic DNA damage rather than reduced DNA damage repair. Our data do not confirm a role for TPPII in the DNA damage response based on nuclear TPPII translocation and p53 stabilization but nevertheless do show increased radiation-induced cell death of selected nontransformed cell types in the absence of the TPPII protease.

The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.

PubMed

Donigan, Katherine A; McLenigan, Mary P; Yang, Wei; Goodman, Myron F; Woodgate, Roger

2014-03-28

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A "steric gate" pol ι mutant is considerably more active in the presence of Mn(2+) compared with Mg(2+) and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be "at risk" for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.

Recognition of DNA abasic site nanocavity by fluorophore-switched probe: Suitable for all sequence environments

NASA Astrophysics Data System (ADS)

Wang, Ying; Hu, Yuehua; Wu, Tao; Zhang, Lihua; Liu, Hua; Zhou, Xiaoshun; Shao, Yong

2016-01-01

Removal of a damaged base in DNA produces an abasic site (AP site) nanocavity. If left un-repaired in vivo by the specific enzyme, this nanocavity will result in nucleotide mutation in the following DNA replication. Therefore, selective recognition of AP site nanocavity by small molecules is important for identification of such DNA damage and development of genetic drugs. In this work, we investigate the fluorescence behavior of isoquinoline alkaloids including palmatine (PAL), berberine (BER), epiberberine (EPI), jatrorrhizine (JAT), coptisine (COP), coralyne (COR), worenine (WOR), berberrubine (BEU), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT) upon binding with the AP nanocavity. PAL is screened out as the most efficient fluorophore-switched probe to recognize the AP nanocavity over the fully matched DNA. Its fluorescence enhancement occurs for all of the AP nanocavity sequence environments, which has not been achieved by the previously used probes. The bridged π conjugation effect should partially contribute to the AP nanocavity-specific fluorescence, as opposed to the solvent effect. Due to the strong binding with the AP nanocavity, PAL will find wide applications in the DNA damage recognition and sensor development.

Parkin in cancer: Mitophagy-related/unrelated tasks.

PubMed

Eid, Nabil; Kondo, Yoichi

2017-03-08

Dysfunctional mitochondria may produce excessive reactive oxygen species, thus inducing DNA damage, which may be oncogenic if not repaired. As a major role of the PINK1-Parkin pathway involves selective autophagic clearance of damaged mitochondria via a process termed mitophagy, Parkin-mediated mitophagy may be a tumor-suppressive mechanism. As an alternative mechanism for tumor inhibition beyond mitophagy, Parkin has been reported to have other oncosuppressive functions such as DNA repair, negative regulation of cell proliferation and stimulation of p53 tumor suppressor function. The authors recently reported that acute ethanol-induced mitophagy in hepatocytes was associated with Parkin mitochondrial translocation and colocalization with accumulated 8-OHdG (a marker of DNA damage and mutagenicity). This finding suggests: (1) the possibility of Parkin-mediated repair of damaged mitochondrial DNA in hepatocytes of ethanol-treated rats (ETRs) as an oncosuppressive mechanism; and (2) potential induction of cytoprotective mitophagy in ETR hepatocytes if mitochondrial damage is too severe to be repaired. Below is a summary of the various roles Parkin plays in tumor suppression, which may or may not be related to mitophagy. A proper understanding of the various tasks performed by Parkin in tumorigenesis may help in cancer therapy by allowing the PINK1-Parkin pathway to be targeted.

Evaluation of imazethapyr-induced DNA oxidative damage by alkaline Endo III- and Fpg-modified single-cell gel electrophoresis assay in Hypsiboas pulchellus tadpoles (Anura, Hylidae).

PubMed

Pérez-Iglesias, Juan Manuel; Ruiz de Arcaute, Celeste; Natale, Guillermo S; Soloneski, S; Larramendy, Marcelo L

2017-08-01

Imazethapyr (IMZT) is a selective postemergent herbicide with residual action. Available data analyzing its effects in aquatic vertebrates are scarce. In previous studies, we demonstrated that IMZT induces lesions into the DNA of Hypsiboas pulchellus tadpoles using the single-cell gel electrophoresis (SCGE) assay as a biomarker for genotoxicity. Currently, this assay can be modified by including incubation with lesion-specific endonucleases, e.g., endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which detect oxidized pyrimidine and purine bases, respectively. The aim of this study was to evaluate the role of oxidative stress in the genotoxic damage in circulating blood cells of H. pulchellus tadpoles exposed to the IMZT-based Pivot H ® formulation (10.59% IMZT) at a concentration equivalent to 25% of the LC 50 (96h) value (0.39mg/L IMZT) during 48 and 96h. Our results demonstrate that the herbicide induces oxidative DNA damage on H. pulchellus tadpoles at purines bases but not at pyrimidines. Our findings represent the first evidence of oxidative damage caused by IMZT on anuran DNA using the alkaline restriction enzyme-modified SCGE assay. Copyright © 2017 Elsevier Inc. All rights reserved.

Genotoxic effects of environmental endocrine disruptors on the aquatic insect Chironomus riparius evaluated using the comet assay.

PubMed

Martínez-Paz, Pedro; Morales, Mónica; Martínez-Guitarte, José Luis; Morcillo, Gloria

2013-12-12

Genotoxicity is one of the most important toxic endpoints in chemical toxicity testing and environmental risk assessment. The aim of this study was to evaluate the genotoxic potential of various environmental pollutants frequently found in aquatic environments and characterized by their endocrine disrupting activity. Monitoring of DNA damage was undertaken after in vivo exposures of the aquatic larvae of the midge Chironomus riparius, a model organism that represents an abundant and ecologically relevant macroinvertebrate, widely used in freshwater toxicology. DNA-induced damage, resulting in DNA fragmentation, was quantified by the comet assay after short (24 h) and long (96 h) exposures to different concentrations of the selected toxicants: bisphenol A (BPA), nonylphenol (NP), pentachlorophenol (PCP), tributyltin (TBT) and triclosan (TCS). All five compounds were found to have genotoxic activity as demonstrated by significant increases in all the comet parameters (%DNA in tail, tail length, tail moment and Olive tail moment) at all tested concentrations. Persistent exposure did not increase the extent of DNA damage, except for TCS at the highest concentration, but generally there was a reduction in DNA damage thought to be associated with the induction of the detoxification processes and repairing mechanisms. Comparative analysis showed differences in the genotoxic potential between the chemicals, as well as significant time and concentration-dependent variations, which most likely reflect differences in the ability to repair DNA damage under the different treatments. The present report demonstrates the sensitivity of the benthic larvae of C. riparius to these environmental genotoxins suggesting its potential as biomonitor organism in freshwater ecosystems. The results obtained about the DNA-damaging potential of these environmental pollutants reinforce the need for additional studies on the genotoxicity of endocrine active substances that, by linking genotoxic activity to other biological responses, could provide further understanding of adverse effects in aquatic environments. Copyright © 2013 Elsevier B.V. All rights reserved.

A single portion of blueberry (Vaccinium corymbosum L) improves protection against DNA damage but not vascular function in healthy male volunteers.

PubMed

Del Bó, Cristian; Riso, Patrizia; Campolo, Jonica; Møller, Peter; Loft, Steffen; Klimis-Zacas, Dorothy; Brambilla, Ada; Rizzolo, Anna; Porrini, Marisa

2013-03-01

It has been suggested that anthocyanin-rich foods may exert antioxidant effects and improve vascular function as demonstrated mainly in vitro and in the animal model. Blueberries are rich sources of anthocyanins and we hypothesized that their intake could improve cell protection against oxidative stress and affect endothelial function in humans. The aim of the study was to investigate the effect of one portion (300 g) of blueberries on selected markers of oxidative stress and antioxidant protection (endogenous and oxidatively induced DNA damage) and of vascular function (changes in peripheral arterial tone and plasma nitric oxide levels) in male subjects. In a randomized cross-over design, separated by a wash out period ten young volunteers received one portion of blueberries ground by blender or one portion of a control jelly. Before and after consumption (at 1, 2, and 24 hours), blood samples were collected and used to evaluate anthocyanin absorption (through mass spectrometry), endogenous and H(2)O(2)-induced DNA damage in blood mononuclear cells (through the comet assay), and plasma nitric oxide concentrations (through a fluorometric assay). Peripheral arterial function was assessed by means of Endo-PAT 2000. Blueberries significantly reduced (P < .01) H(2)O(2)-induced DNA damage (-18%) 1 hour after blueberry consumption compared to control. No significant differences were observed for endogenous DNA damage, peripheral arterial function and nitric oxide levels after blueberry intake. In conclusion, one portion of blueberries seems sufficient to improve cell antioxidant defense against DNA damage, but further studies are necessary to understand their role on vascular function. Copyright © 2013 Elsevier Inc. All rights reserved.

Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

PubMed

Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-02-04

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Age-dependent systemic DNA damage in early Type 2 Diabetes mellitus.

PubMed

Rogulj, Dinko; El Aklouk, Ismail; Konjevoda, Paško; Ljubić, Spomenka; Pibernik Okanović, Mirjana; Barbir, Ante; Luburić, Marijana; Radman, Maja; Budinski, Ninoslav; Vučić Lovrenčić, Marijana

2017-01-01

Oxidative stress, capable of eliciting damage to various biomolecules including DNA, is a recognized component of diabetes mellitus and its complications. Metabolic syndrome (MetS) is associated with the development of type 2 diabetes mellitus (T2DM), as well as other unfavorable outcomes. The aim of this study was to elucidate the role of oxidative stress in the development of T2DM, by investigating association of oxidative DNA damage with metabolic parameters in subjects with MetS and early T2DM. Selected anthropometric and biochemical parameters of MetS, inflammation and oxidative DNA damage: body mass index (BMI), fatty liver index (FLI), waist circumference (WC), total cholesterol, HDL and LDL-cholesterol, gamma-glutamyl transpeptidase (GGT), uric acid, C-reactive protein (CRP), total leukocyte/neutrophil count, and urinary 8-hidroxy-deoxyguanosine (u-8-OHdG) were assessed in male subjects with MetS and both younger (≤55 years) and older (>55 years) subjects with T2DM of short duration without complications. BMI, FLI, WC, total and LDL-cholesterol and uric acid were higher, while the u-8-OHdG was lower in MetS group, when compared to older T2DM subjects. None of these parameters were different neither between MetS and younger T2DM, nor between two sub-groups of subjects with T2DM. Values of CRP, HDL-cholesterol, triglycerides, GGT, leukocytes and neutrophils were not different between all examined groups of subjects. Higher 8-OHdG in older subjects with T2DM suggests that both aging process and diabetes could contribute to the development of DNA damage. Oxidative DNA damage cannot serve as an universal early marker of T2DM.

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH).

PubMed

Cortés-Gutiérrez, Elva I; Ortíz-Hernández, Brenda L; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

2013-02-19

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

Elevated Rate of Genome Rearrangements in Radiation-Resistant Bacteria.

PubMed

Repar, Jelena; Supek, Fran; Klanjscek, Tin; Warnecke, Tobias; Zahradka, Ksenija; Zahradka, Davor

2017-04-01

A number of bacterial, archaeal, and eukaryotic species are known for their resistance to ionizing radiation. One of the challenges these species face is a potent environmental source of DNA double-strand breaks, potential drivers of genome structure evolution. Efficient and accurate DNA double-strand break repair systems have been demonstrated in several unrelated radiation-resistant species and are putative adaptations to the DNA damaging environment. Such adaptations are expected to compensate for the genome-destabilizing effect of environmental DNA damage and may be expected to result in a more conserved gene order in radiation-resistant species. However, here we show that rates of genome rearrangements, measured as loss of gene order conservation with time, are higher in radiation-resistant species in multiple, phylogenetically independent groups of bacteria. Comparison of indicators of selection for genome organization between radiation-resistant and phylogenetically matched, nonresistant species argues against tolerance to disruption of genome structure as a strategy for radiation resistance. Interestingly, an important mechanism affecting genome rearrangements in prokaryotes, the symmetrical inversions around the origin of DNA replication, shapes genome structure of both radiation-resistant and nonresistant species. In conclusion, the opposing effects of environmental DNA damage and DNA repair result in elevated rates of genome rearrangements in radiation-resistant bacteria. Copyright © 2017 Repar et al.

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

PubMed Central

Wang, Shu-Huei; Lin, Pei-Ya; Chiu, Ya-Chen; Huang, Ju-Sui; Kuo, Yi-Tsen; Wu, Jen-Chine; Chen, Chin-Chuan

2015-01-01

Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity. PMID:26218133

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

PubMed

Patel, S; Sprung, A U; Keller, B A; Heaton, V J; Fisher, L M

1997-10-01

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells.

PubMed

Bailis, Julie M; Weidmann, Alyson G; Mariano, Natalie F; Barton, Jacqueline K

2017-07-03

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)] 2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

Recruitment of TRF2 to laser-induced DNA damage sites.

PubMed

Huda, Nazmul; Abe, Satoshi; Gu, Ling; Mendonca, Marc S; Mohanty, Samarendra; Gilley, David

2012-09-01

Several lines of evidence suggest that the telomere-associated protein TRF2 plays critical roles in the DNA damage response. TRF2 is rapidly and transiently phosphorylated by an ATM-dependent pathway in response to DNA damage and this DNA damage-induced phosphoryation is essential for the DNA-PK-dependent pathway of DNA double-strand break repair (DSB). However, the type of DNA damage that induces TRF2 localization to the damage sites, the requirement for DNA damage-induced phosphorylation of TRF2 for its recruitment, as well as the detailed kinetics of TRF2 accumulation at DNA damage sites have not been fully investigated. In order to address these questions, we used an ultrafast femtosecond multiphoton laser and a continuous wave 405-nm single photon laser to induce DNA damage at defined nuclear locations. Our results showed that DNA damage produced by a femtosecond multiphoton laser was sufficient for localization of TRF2 to these DNA damage sites. We also demonstrate that ectopically expressed TRF2 was recruited to DNA lesions created by a 405-nm laser. Our data suggest that ATM and DNA-PKcs kinases are not required for TRF2 localization to DNA damage sites. Furthermore, we found that phosphorylation of TRF2 at residue T188 was not essential for its recruitment to laser-induced DNA damage sites. Thus, we provide further evidence that a protein known to function in telomere maintenance, TRF2, is recruited to sites of DNA damage and plays critical roles in the DNA damage response. Copyright © 2012 Elsevier Inc. All rights reserved.

Photosensitization of DNA damage by a new cationic pyropheophorbide derivative: sequence-specific formation of a frank scission.

PubMed

Kanony, Claire; Fabiano-Tixier, Anne-Sylvie; Ravanat, Jean-Luc; Vicendo, Patricia; Paillous, Nicole

2003-06-01

Pyropheophorbides are red-absorbing porphyrin-like photosensitizers that may interact with DNA either by intercalation or by external binding with self-stacking according to the value of the nucleotide to chromophore molar ratio (N/C). This article reports on the nature and sequence selectivity of the DNA damage photoinduced by a water-soluble chlorhydrate of aminopyropheophorbide. First, this pyropheophorbide is shown to induce on irradiation the cleavage of phiX174 DNA by both Type-I and -II mechanisms, suggested by scavengers and D2O effects. These conclusions are then improved by sequencing experiments performed on a 20-mer oligodeoxynucleotide (ODN) irradiated at wavelengths >345 nm in the presence of the dye, N/C varying from 2.5 to 0.5. Oxidation of all guanine residues to the same extent is observed after piperidine treatment on both single- and double-stranded ODN. Moreover, unexpectedly, a remarkable sequence-selective cleavage occurring at a 5'-CG-3' site is detected before alkali treatment. This frank break is clearly predominant for a low nucleotide to chromophore molar ratio, corresponding to a self-stacking of the dye along the DNA helix. The electrophoretic properties of the band suggest that this lesion results from a sugar oxidation, which leads via a base release to a ribonolactone residue. The proposal is supported by high-performance liquid chromatography-matrix-assisted laser desorption-ionization mass spectrometry experiments that also reveal other sequence-selective frank scissions of lower intensity at 5'-GC-3' or other 5'-CG-3' sites. This sequence selectivity is discussed with regard to the binding selectivity of cationic porphyrins.

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

PubMed

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells.

PubMed

Cho, Eun-Ah; Juhnn, Yong-Sung

2012-06-01

Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

11th International Conference of Radiation Research

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1999-07-18

Topics discussed in the conference included the following: Radiation Physics, Radiation Chemistry and modelling--Radiation physics and dosimetry; Electron transfer in biological media; Radiation chemistry; Biophysical and biochemical modelling; Mechanisms of DNA damage; Assays of DNA damage; Energy deposition in micro volumes; Photo-effects; Special techniques and technologies; Oxidative damage. Molecular and cellular effects-- Photobiology; Cell cycle effects; DNA damage: Strand breaks; DNA damage: Bases; DNA damage Non-targeted; DNA damage: other; Chromosome aberrations: clonal; Chromosomal aberrations: non-clonal; Interactions: Heat/Radiation/Drugs; Biochemical effects; Protein expression; Gene induction; Co-operative effects; ``Bystander'' effects; Oxidative stress effects; Recovery from radiation damage. DNA damage and repair -- DNAmore » repair genes; DNA repair deficient diseases; DNA repair enzymology; Epigenetic effects on repair; and Ataxia and ATM.« less

The Steric Gate of DNA Polymerase ι Regulates Ribonucleotide Incorporation and Deoxyribonucleotide Fidelity*

PubMed Central

Donigan, Katherine A.; McLenigan, Mary P.; Yang, Wei; Goodman, Myron F.; Woodgate, Roger

2014-01-01

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A “steric gate” pol ι mutant is considerably more active in the presence of Mn2+ compared with Mg2+ and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be “at risk” for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations. PMID:24532793

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

PubMed

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

2017-04-11

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

PubMed Central

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

2017-01-01

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles. PMID:28398335

Tyrosyl-DNA phosphodiesterase inhibitors: Progress and potential.

PubMed

Laev, Sergey S; Salakhutdinov, Nariman F; Lavrik, Olga I

2016-11-01

DNA topoisomerases are essential during transcription and replication. The therapeutic mechanism of action of topoisomerase inhibitors is enzyme poisoning rather than catalytic inhibition. Tyrosyl-DNA phosphodiesterases 1 or 2 were found as DNA repair enzymes hydrolyzing the covalent bond between the tyrosyl residue of topoisomerases I or II and the 3'- or 5'-phosphate groups in DNA, respectively. Tyrosyl-DNA phosphodiesterase 1 is a key enzyme in DNA repair machinery and a promising target for antitumor and neurodegenerative therapy. Inhibitors of tyrosyl-DNA phosphodiesterase 1 could act synergistically with topoisomerase I inhibitors and thereby potentiate the effects of topoisomerase I poisons. Tyrosyl-DNA phosphodiesterase 2 is an enzyme that specifically repairs DNA damages induced by topoisomerase II poisons and causes resistance to these drugs. Selective inhibition of tyrosyl-DNA phosphodiesterase 2 may be a novel approach to overcome intrinsic or acquired resistance to topoisomerase II-targeted drug therapy. Thus, agents that inhibit tyrosyl-DNA phosphodiesterases 1 and 2 have many applications in biochemical and physiological research and they have the potential to become anticancer and antiviral drugs. The structures, mechanism of action and therapeutic rationale of tyrosyl-DNA phosphodiesterase inhibitors and their development for combinations with topoisomerase inhibitors and DNA damaging agents are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Eun-Ah; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

2012-06-01

Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNAmore » repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.« less

In vitro characterization of pol I*, an SOS inducible, error-prone from of Escherichia coli DNA polymerase I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodes, C.S.

E. coli strains carrying mutations in the SOS regulon were screened for the presence of pol I*. Use of pol I* in assays of polymerase fidelity and selectivity has been limited by the low concentration and purity of the enzyme. Therefore, attempts were made to further concentrate and purify pol I*. The template selectivity of pol I* was compared to that of pol I using three models of damaged DNA. UV-irradiated M13 DNA was used in a two-stage termination reaction to determine if pol I* could bypass putative pyrimidine dimers to a greater extent than pol I. In the gelmore » system no reproducibly significant bypass could be detected by either pol I* or pol I. However, the degree of replication by pol I* utilizing UV-irradiated M13 DNA template, was up to 5-fold greater than for pol I. OsO{sub 4}-oxidized M13 DNA was used as a model substrate for oxidative DNA damage. Opposite this substrate incorporation by pol I* is less inhibited than incorporation by pol I. However, in a test of nucleotide selectivity neither pol I*, pol I, nor terminal deoxyribonucleotidyl transferase will incorporate ({alpha}-{sup 32}P)thymine glycol deoxyribonucleotide. The activity of pol I* was compared to that of pol I on the synthetic templates, poly (dA) and poly((dA)+2-AP). Pol I* misincorporated both dCMP and dGMP to a greater extent than pol I when utilizing templates containing 2-aminopurine deoxyribonucleotide.« less

DNA Repair and Genome Maintenance in Bacillus subtilis

PubMed Central

Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

2012-01-01

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

PubMed

Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

2011-10-01

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Divergence in DNA photorepair efficiency among genotypes from contrasting UV radiation environments in nature.

PubMed

Miner, Brooks E; Kulling, Paige M; Beer, Karlyn D; Kerr, Benjamin

2015-12-01

Populations of organisms routinely face abiotic selection pressures, and a central goal of evolutionary biology is to understand the mechanistic underpinnings of adaptive phenotypes. Ultraviolet radiation (UVR) is one of earth's most pervasive environmental stressors, potentially damaging DNA in any organism exposed to solar radiation. We explored mechanisms underlying differential survival following UVR exposure in genotypes of the water flea Daphnia melanica derived from natural ponds of differing UVR intensity. The UVR tolerance of a D. melanica genotype from a high-UVR habitat depended on the presence of visible and UV-A light wavelengths necessary for photoenzymatic repair of DNA damage, a repair pathway widely shared across the tree of life. We then measured the acquisition and repair of cyclobutane pyrimidine dimers, the primary form of UVR-caused DNA damage, in D. melanica DNA following experimental UVR exposure. We demonstrate that genotypes from high-UVR habitats repair DNA damage faster than genotypes from low-UVR habitats in the presence of visible and UV-A radiation necessary for photoenzymatic repair, but not in dark treatments. Because differences in repair rate only occurred in the presence of visible and UV-A radiation, we conclude that differing rates of DNA repair, and therefore differential UVR tolerance, are a consequence of variation in photoenzymatic repair efficiency. We then rule out a simple gene expression hypothesis for the molecular basis of differing repair efficiency, as expression of the CPD photolyase gene photorepair did not differ among D. melanica lineages, in both the presence and absence of UVR. © 2015 John Wiley & Sons Ltd.

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

PubMed

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

Oxidative DNA damage in prostate cancer patients consuming tomato sauce-based entrees as a whole-food intervention.

PubMed

Chen, L; Stacewicz-Sapuntzakis, M; Duncan, C; Sharifi, R; Ghosh, L; van Breemen, R; Ashton, D; Bowen, P E

2001-12-19

Human prostate tissues are vulnerable to oxidative DNA damage. The risk of prostate cancer is lower in men reporting higher consumption of tomato products, which contain high levels of the antioxidant lycopene. We examined the effects of consumption of tomato sauce-based pasta dishes on lycopene uptake, oxidative DNA damage, and prostate-specific antigen (PSA) levels in patients already diagnosed with prostate cancer. Thirty-two patients with localized prostate adenocarcinoma consumed tomato sauce-based pasta dishes for the 3 weeks (30 mg of lycopene per day) preceding their scheduled radical prostatectomy. Serum and prostate lycopene concentrations, serum PSA levels, and leukocyte DNA oxidative damage (ratio of 8-hydroxy-2'-deoxyguanosine [8-OHdG] to 2'-deoxyguanosine [dG]) were assessed before and after the dietary intervention. DNA oxidative damage was assessed in resected prostate tissue from study participants and from seven randomly selected prostate cancer patients. All statistical tests were two-sided. After the dietary intervention, serum and prostate lycopene concentrations were statistically significantly increased, from 638 nM (95% confidence interval [CI] = 512 to 764 nM) to 1258 nM (95% CI = 1061 to 1455 nM) (P<.001) and from 0.28 nmol/g (95% CI = 0.18 to 0.37 nmol/g) to 0.82 nmol/g (95% CI = 0.57 to 1.11 nmol/g) (P <.001), respectively. Compared with preintervention levels, leukocyte oxidative DNA damage was statistically significantly reduced after the intervention, from 0.61 8-OHdG/10(5) dG (95% CI = 0.45 to 0.77 8-OHdG/10(5) dG) to 0.48 8-OHdG/ 10(5) dG (95% CI = 0.41 to 0.56 8-OHdG/10(5) dG) (P =.005). Furthermore, prostate tissue oxidative DNA damage was also statistically significantly lower in men who had the intervention (0.76 8-OHdG/10(5) dG [95% CI = 0.55 to 0.96 8-OHdG/10(5) dG]) than in the randomly selected patients (1.06 8-OHdG/10(5) dG [95% CI = 0.62 to 1.51 8-OHdG/10(5) dG]; P =.03). Serum PSA levels decreased after the intervention, from 10.9 ng/mL (95% CI = 8.7 to 13.2 ng/mL) to 8.7 ng/mL (95% CI = 6.8 to 10.6 ng/mL) (P<.001). These data indicate a possible role for a tomato sauce constituent, possibly lycopene, in the treatment of prostate cancer and warrant further testing with a larger sample of patients, including a control group.

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

PubMed

Bist, Itti; Bhakta, Snehasis; Jiang, Di; Keyes, Tia E; Martin, Aaron; Forster, Robert J; Rusling, James F

2017-11-21

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S N 2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu 2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10 6 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD 50 and Comet assay results.

Selective sensitization to DNA-damaging agents in a human rhabdomyosarcoma cell line with inducible wild-type p53 overexpression.

PubMed

Gibson, A A; Harwood, F G; Tillman, D M; Houghton, J A

1998-01-01

Drug-induced cytotoxicity or apoptosis may be influenced by the expression of the p53 tumor suppressor gene and by the specific oncogene expressed, which may dictate the threshold at which a cytotoxic response may by induced. The objective of the study was to elucidate how DNA-damaging agents with different mechanisms of action were sensitized in the context of expression of the Pax3/FKHR fusion protein, a transformation event unique to alveolar rhabdomyosarcomas (ARMSs), and wild-type p53 (wtp53). A wtp53 cDNA was subcloned into the pGRE5-2/EBV vector with dexamethasone-inducible overexpression and transfected into Rh30 ARMS cells that express Pax3/FKHR and a mutant p53 phenotype. Following dexamethasone induction of wtp53 overexpression in a derived clone (Cl.#27), growth was slowed, and cells accumulated in G1. Functional wtp53 activity was demonstrated by selective transactivation of p50-2, a wtp53 chloramphenicol acetyltransferase reporter construct, and by up-regulated expression of endogenous p21Waf1. Data demonstrated p53-dependent sensitization (> or = 4-fold) to bleomycin, actinomycin D, and 5-fluorouracil and considerably less p53-dependence (< or = 2-fold) for doxorubicin, topotecan, etoposide, and cisplatin in Cl.#27 compared to an equivalent clone containing the pGRE5-EBV vector alone (VC#3). Data demonstrate that ARMS cells show a selective sensitization to DNA-damaging agents when wtp53 is overexpressed. The cytotoxic activity of agents that are not potentiated substantially must, therefore, depend upon p53-independent factors that relate to the mechanism of drug action.

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawiak, Anna; Piosik, Jacek; Stasilojc, Grzegorz

2007-09-15

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topomore » II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.« less

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

PubMed

Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

2014-06-01

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchetti, Francesco; Bishop, Jack; Gingerich, John

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widelymore » used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. In conclusion, these findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.« less

Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair

DOE PAGES

Marchetti, Francesco; Bishop, Jack; Gingerich, John; ...

2015-01-08

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widelymore » used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. In conclusion, these findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.« less

DNA damage and oxidative stress response to selenium yeast in the non-smoking individuals: a short-term supplementation trial with respect to GPX1 and SEPP1 polymorphism.

PubMed

Jablonska, E; Raimondi, S; Gromadzinska, J; Reszka, E; Wieczorek, E; Krol, M B; Smok-Pieniazek, A; Nocun, M; Stepnik, M; Socha, K; Borawska, M H; Wasowicz, W

2016-12-01

Selenium, both essential and toxic element, is considered to protect against cancer, though human supplementation trials have generated many inconsistent data. Genetic background may partially explain a great variability of the studies related to selenium and human health. The aim of this study was to assess whether functional polymorphisms within two selenoprotein-encoding genes modify the response to selenium at the level of oxidative stress, DNA damage, and mRNA expression, especially in the individuals with a relatively low selenium status. The trial involved 95 non-smoking individuals, stratified according to GPX1 rs1050450 and SEPP1 rs3877899 genotypes, and supplemented with selenium yeast (200 µg) for 6 weeks. Blood was collected at four time points, including 4 weeks of washout. After genotype stratification, the effect of GPX1 rs1050450 on lower GPx1 activity responsiveness was confirmed; however, in terms of DNA damage, we failed to indicate that individuals homozygous for variant allele may especially benefit from the increased selenium intake. Surprisingly, considering gene and time interaction, GPX1 polymorphism was observed to modify the level of DNA strand breaks during washout, showing a significant increase in GPX1 wild-type homozygotes. Regardless of the genotype, selenium supplementation was associated with a selectively suppressed selenoprotein mRNA expression and inconsistent changes in oxidative stress response, indicating for overlapped, antioxidant, and prooxidant effects. Intriguingly, DNA damage was not influenced by supplementation, but it was significantly increased during washout. These results point to an unclear relationship between selenium, genotype, and DNA damage.

Protein Interactions in T7 DNA Replisome Facilitate DNA Damage Bypass.

PubMed

Zou, Zhenyu; Chen, Ze; Xue, Qizhen; Xu, Ying; Xiong, Jingyuan; Yang, Ping; Le, Shuai; Zhang, Huidong

2018-06-14

DNA replisome inevitably encounters DNA damage during DNA replication. T7 DNA replisome contains DNA polymerase (gp5), the processivity factor thioredoxin (trx), helicase-primase (gp4), and ssDNA binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated the strand-displacement DNA synthesis past 8-oxoG or O6-MeG at the synthetic DNA fork by T7 DNA replisome. DNA damage does not obviously affect the binding affinities among helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6-MeG, as well as GC-rich template sequence clusters, inhibit the strand-displacement DNA synthesis and produce partial extension products. Relative to gp4 ΔC-tail, gp4 promotes the DNA damage bypass. The presence of gp2.5 further promotes this bypass. Thus, the interactions of polymerase with helicase and ssDNA binidng protein faciliate the DNA damage bypass. Similarly, accessory proteins in other complicated DNA replisomes also facilitate the DNA damage bypass. This work provides the novel mechanism information of DNA damage bypass by DNA replisome. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of γ-radiation-induced DNA damage in two species of bivalves and their relative sensitivity using comet assay.

PubMed

Praveen Kumar, M K; Shyama, S K; Sonaye, B S; Naik, U Roshini; Kadam, S B; Bipin, P D; D'costa, A; Chaubey, R C

2014-05-01

Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of 'Comet assay' for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in both bivalve species. This showed a dose-dependent increase of genetic damage induced in bivalves by EMS as well as gamma radiation. Further, the highest DNA damage was observed at 24h. The damage gradually decreased with time, i.e. was smaller at 48 and 72 h than at 24h post irradiation in both species of bivalves. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the post irradiation time advanced. The present study reveals that gamma radiation induces single strand breaks in DNA as measured by alkaline comet assay in bivalves and comet assay serves as a sensitive and rapid method to detect genotoxicity of gamma radiation. This study further indicates that both M. casta and P. malabarica exhibit almost identical sensitivity to gamma radiation as measured by DNA damage. Copyright © 2014 Elsevier B.V. All rights reserved.

"Gadd45b" Knockout Mice Exhibit Selective Deficits in Hippocampus-Dependent Long-Term Memory

ERIC Educational Resources Information Center

Leach, Prescott T.; Poplawski, Shane G.; Kenney, Justin W.; Hoffman, Barbara; Liebermann, Dan A.; Abel, Ted; Gould, Thomas J.

2012-01-01

Growth arrest and DNA damage-inducible [beta] ("Gadd45b") has been shown to be involved in DNA demethylation and may be important for cognitive processes. "Gadd45b" is abnormally expressed in subjects with autism and psychosis, two disorders associated with cognitive deficits. Furthermore, several high-throughput screens have identified "Gadd45b"…

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

PubMed

Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

2017-03-01

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

Making Sense of Missense in the Lynch Syndrome: The Clinical Perspective

PubMed Central

Lynch, Henry T.; Jascur, Thomas; Lanspa, Stephen; Boland, C. Richard

2010-01-01

The DNA mismatch repair system provides critical genetic housekeeping, and its failure is associated with tumorigenesis. Through distinct domains on the DNA mismatch repair proteins, the system recognizes and repairs errors occurring during DNA synthesis, but signals apoptosis when the DNA damage cannot be repaired. Certain missense mutations in the mismatch repair genes can selectively alter just one of these functions. This impacts the clinical features of tumors associated with defective DNA mismatch repair activity. New work reported by Xie et al. in this issue of the journal (beginning on page XXX) adds to the understanding of DNA mismatch repair. PMID:20978117

The comet assay as a tool for human biomonitoring studies: the ComNet project.

PubMed

Collins, Andrew; Koppen, Gudrun; Valdiglesias, Vanessa; Dusinska, Maria; Kruszewski, Marcin; Møller, Peter; Rojas, Emilio; Dhawan, Alok; Benzie, Iris; Coskun, Erdem; Moretti, Massimo; Speit, Günter; Bonassi, Stefano

2014-01-01

The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view. Copyright © 2013 Elsevier B.V. All rights reserved.

Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa.

PubMed

Ebner, T; Shebl, O; Moser, M; Mayer, R B; Arzt, W; Tews, G

2011-01-01

Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Interplay between DNA repair and inflammation, and the link to cancer

PubMed Central

Kidane, Dawit; Chae, Wook Jin; Czochor, Jennifer; Eckert, Kristin A.; Glazer, Peter M.; Bothwell, Alfred L. M.; Sweasy, Joann B.

2015-01-01

DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer. Reactive oxygen and nitrogen species (RONs) produced by inflammatory cells at sites of infection can induce DNA damage. RONs can also amplify inflammatory responses, leading to increased DNA damage. Here, we focus on the links between DNA damage, repair, and inflammation, as they relate to cancer. We examine the interplay between chronic inflammation, DNA damage and repair and review recent findings in this rapidly emerging field, including the links between DNA damage and the innate immune system, and the roles of inflammation in altering the microbiome, which subsequently leads to the induction of DNA damage in the colon. Mouse models of defective DNA repair and inflammatory control are extensively reviewed, including treatment of mouse models with pathogens, which leads to DNA damage. The roles of microRNAs in regulating inflammation and DNA repair are discussed. Importantly, DNA repair and inflammation are linked in many important ways, and in some cases balance each other to maintain homeostasis. The failure to repair DNA damage or to control inflammatory responses has the potential to lead to cancer. PMID:24410153

Mitochondrial DNA damage is associated with damage accrual and disease duration in patients with Systemic Lupus Erythematosus

PubMed Central

López-López, Linnette; Nieves-Plaza, Mariely; Castro, María del R.; Font, Yvonne M.; Torres-Ramos, Carlos; Vilá, Luis M.; Ayala-Peña, Sylvette

2014-01-01

Objective To determine the extent of mitochondrial DNA (mtDNA) damage in systemic lupus erythematosus (SLE) patients compared to healthy subjects and to determine the factors associated with mtDNA damage among SLE patients. Methods A cross-sectional study was performed in 86 SLE patients (per American College of Rheumatology classification criteria) and 86 healthy individuals matched for age and gender. Peripheral blood mononuclear cells (PBMCs) were collected from subjects to assess the relative amounts of mtDNA damage. Quantitative polymerase chain reaction assay was used to measure the frequency of mtDNA lesions and mtDNA abundance. Socioeconomic-demographic features, clinical manifestations, pharmacologic treatment, disease activity, and damage accrual were determined. Statistical analyses were performed using t test, pairwise correlation, and Pearson’s chi-square test (or Fisher’s exact test) as appropriate. Results Among SLE patients, 93.0% were women. The mean (SD) age was 38.0 (10.4) years and the mean (SD) disease duration was 8.7 (7.5) years. SLE patients exhibited increased levels of mtDNA damage as shown by higher levels of mtDNA lesions and decreased mtDNA abundance as compared to healthy individuals. There was a negative correlation between disease damage and mtDNA abundance and a positive correlation between mtDNA lesions and disease duration. No association was found between disease activity and mtDNA damage. Conclusion PBMCs from SLE patients exhibited more mtDNA damage compared to healthy subjects. Higher levels of mtDNA damage were observed among SLE patients with major organ involvement and damage accrual. These results suggest that mtDNA damage have a potential role in the pathogenesis of SLE. PMID:24899636

Mitochondrial DNA damage is associated with damage accrual and disease duration in patients with systemic lupus erythematosus.

PubMed

López-López, L; Nieves-Plaza, M; Castro, M del R; Font, Y M; Torres-Ramos, C A; Vilá, L M; Ayala-Peña, S

2014-10-01

To determine the extent of mitochondrial DNA (mtDNA) damage in systemic lupus erythematosus (SLE) patients compared to healthy subjects and to determine the factors associated with mtDNA damage among SLE patients. A cross-sectional study was performed in 86 SLE patients (per American College of Rheumatology classification criteria) and 86 healthy individuals matched for age and gender. Peripheral blood mononuclear cells (PBMCs) were collected from subjects to assess the relative amounts of mtDNA damage. Quantitative polymerase chain reaction assay was used to measure the frequency of mtDNA lesions and mtDNA abundance. Socioeconomic-demographic features, clinical manifestations, pharmacologic treatment, disease activity, and damage accrual were determined. Statistical analyses were performed using t test, pairwise correlation, and Pearson's chi-square test (or Fisher's exact test) as appropriate. Among SLE patients, 93.0% were women. The mean (SD) age was 38.0 (10.4) years and the mean (SD) disease duration was 8.7 (7.5) years. SLE patients exhibited increased levels of mtDNA damage as shown by higher levels of mtDNA lesions and decreased mtDNA abundance as compared to healthy individuals. There was a negative correlation between disease damage and mtDNA abundance and a positive correlation between mtDNA lesions and disease duration. No association was found between disease activity and mtDNA damage. PBMCs from SLE patients exhibited more mtDNA damage compared to healthy subjects. Higher levels of mtDNA damage were observed among SLE patients with major organ involvement and damage accrual. These results suggest that mtDNA damage have a potential role in the pathogenesis of SLE. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

PubMed

Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

2011-06-24

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Types and Consequences of DNA Damage

EPA Science Inventory

This review provides a concise overview of the types of DNA damage and the molecular mechanisms by which a cell senses DNA damage, repairs the damage, converts the damage into a mutation, or dies as a consequence of unrepaired DNA damage. Such information is important in consid...

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

PubMed Central

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study. PMID:26862534

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines.

PubMed

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study.

Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder.

PubMed

Czarny, Piotr; Kwiatkowski, Dominik; Toma, Monika; Gałecki, Piotr; Orzechowska, Agata; Bobińska, Kinga; Bielecka-Kowalska, Anna; Szemraj, Janusz; Berk, Michael; Anderson, George; Śliwiński, Tomasz

2016-11-20

BACKGROUND Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. MATERIAL AND METHODS Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). RESULTS We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. CONCLUSIONS The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease.

Single-Nucleotide Polymorphisms of Genes Involved in Repair of Oxidative DNA Damage and the Risk of Recurrent Depressive Disorder

PubMed Central

Czarny, Piotr; Kwiatkowski, Dominik; Toma, Monika; Gałecki, Piotr; Orzechowska, Agata; Bobińska, Kinga; Bielecka-Kowalska, Anna; Szemraj, Janusz; Berk, Michael; Anderson, George; Śliwiński, Tomasz

2016-01-01

Background Depressive disorder, including recurrent type (rDD), is accompanied by increased oxidative stress and activation of inflammatory pathways, which may induce DNA damage. This thesis is supported by the presence of increased levels of DNA damage in depressed patients. Such DNA damage is repaired by the base excision repair (BER) pathway. BER efficiency may be influenced by polymorphisms in BER-related genes. Therefore, we genotyped nine single-nucleotide polymorphisms (SNPs) in six genes encoding BER proteins. Material/Methods Using TaqMan, we selected and genotyped the following SNPs: c.-441G>A (rs174538) of FEN1, c.2285T>C (rs1136410) of PARP1, c.580C>T (rs1799782) and c.1196A>G (rs25487) of XRCC1, c.*83A>C (rs4796030) and c.*50C>T (rs1052536) of LIG3, c.-7C>T (rs20579) of LIG1, and c.-468T>G (rs1760944) and c.444T>G (rs1130409) of APEX1 in 599 samples (288 rDD patients and 311 controls). Results We found a strong correlation between rDD and both SNPs of LIG3, their haplotypes, as well as a weaker association with the c.-468T>G of APEXI which diminished after Nyholt correction. Polymorphisms of LIG3 were also associated with early onset versus late onset depression, whereas the c.-468T>G polymorphism showed the opposite association. Conclusions The SNPs of genes involved in the repair of oxidative DNA damage may modulate rDD risk. Since this is an exploratory study, the results should to be treated with caution and further work needs to be done to elucidate the exact involvement of DNA damage and repair mechanisms in the development of this disease. PMID:27866211

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

PubMed

Kik, Krzysztof; Studzian, Kazimierz; Wasowska-Łukawska, Małgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-01-01

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

Genetic polymorphisms in 19q13.3 genes associated with alteration of repair capacity to BPDE-DNA adducts in primary cultured lymphocytes.

PubMed

Xiao, Mingyang; Xiao, Sha; Straaten, Tahar van der; Xue, Ping; Zhang, Guopei; Zheng, Xiao; Zhang, Qianye; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Zhu, Guolian; Lu, Xiaobo

2016-12-01

Benzo[a]pyrene(B[a]P), and its ultimate metabolite Benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), are classic DNA damaging carcinogens. DNA damage in cells caused by BPDE is normally repaired by Nucleotide Excision Repair (NER) and Base Excision Repair (BER). Genetic variations in NER and BER can change individual DNA repair capacity to DNA damage induced by BPDE. In the present study we determined the number of in vitro induced BPDE-DNA adducts in lymphocytes, to reflect individual susceptibility to Polycyclic aromatic hydrocarbons (PAHs)-induced carcinogenesis. The BPDE-DNA adduct level in lymphocytes were assessed by high performance liquid chromatography (HPLC) in 281 randomly selected participants. We genotyped for 9 single nucleotide polymorphisms (SNPs) in genes involved in NER (XPB rs4150441, XPC rs2228001, rs2279017 and XPF rs4781560), BER (XRCC1 rs25487, rs25489 and rs1799782) and genes located on chromosome 19q13.2-3 (PPP1R13L rs1005165 and CAST rs967591). We found that 3 polymorphisms in chromosome 19q13.2-3 were associated with lower levels of BPDE-DNA adducts (MinorT allele in XRCC1 rs1799782, minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571). In addition, a modified comet assay was performed to further confirm the above conclusions. We found both minor T allele in PPP1R13L rs1005165 and minor A allele in CAST rs967571 were associated with the lower levels of BPDE-adducts. Our data suggested that the variant genotypes of genes in chromosome 19q13.2-3 are associated with the alteration of repair efficiency to DNA damage caused by Benzo[a]pyrene, and may contribute to enhance predictive value for individual's DNA repair capacity in response to environmental carcinogens. Copyright © 2016 Elsevier B.V. All rights reserved.

XPF expression correlates with clinical outcome in squamous cell carcinoma of the head and neck

PubMed Central

Vaezi, Alec; Wang, XiaoZhe; Buch, Shama; Gooding, William; Wang, Lin; Seethala, Raja R.; Weaver, David T.; D’Andrea, Alan D.; Argiris, Athanassios; Romkes, Marjorie; Niedernhofer, Laura J.; Grandis, Jennifer R.

2011-01-01

Purpose Tumor-specific biomarkers that predict resistance to DNA damaging agents may improve therapeutic outcomes by guiding the selection of effective therapies and limiting morbidity related to ineffective approaches. XPF (ERCC4) is an essential component of several DNA repair pathways and XPF-deficient cells are exquisitely sensitive to DNA damaging agents. The purpose of this study was to determine whether XPF expression levels predict clinical response to DNA damaging agents in head and neck squamous cell carcinoma (HNSCC). Experimental Design Quantitative immunohistochemistry was used to measure XPF expression in tumors from a cohort of 80 patients with newly diagnosed HNSCC treated with radiation therapy with or without platinum-based chemotherapy; samples were collected prospectively. Genomic DNA isolated from blood samples was analyzed for nine single nucleotide polymorphisms in the XPF gene using a custom array. The primary endpoint was progression-free survival (PFS). Results XPF expression was higher in tumors from the oral cavity than from the other sites (p<0.01). High XPF expression correlated with early time to progression both by univariate (HR =1.87, p=0.03) and multivariate analysis (HR =1.83, p=0.05). The one year PFS for high expressers was 47% (95% CI = 31% – 62%) compared to 72% (95% CI = 55% – 83%) for low expressers. In addition, we identified four XPF single nucleotide polymorphisms (SNPs) that demonstrated marginal association with treatment failure. Conclusions Expression level of XPF in HNSCC tumors correlates with clinical response to DNA damaging agents. XPF has potential to guide next-generation personalized cancer therapy. PMID:21737503

Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response.

PubMed

Luo, Man; Bao, Zhengqiang; Xu, Feng; Wang, Xiaohui; Li, Fei; Li, Wen; Chen, Zhihua; Ying, Songmin; Shen, Huahao

2018-04-14

The inflammatory cascade can be initiated with the recognition of damaged DNA. Macrophages play an essential role in particulate matter (PM)-induced airway inflammation. In this study, we aim to explore the PM induced DNA damage response of macrophages and its function in airway inflammation. The DNA damage response and inflammatory response were assessed using bone marrow-derived macrophages following PM treatment and mouse model instilled intratracheally with PM. We found that PM induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response, represented by nuclear RPA, 53BP1 and γH2AX foci formation. Genetic ablation or chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcl1, Cxcl2 and Ifn-γ after PM stimulation in bone marrow-derived macrophages. Similar to that seen in vitro , mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the PM challenge, suggesting a protective role of DNA damage sensor during inflammation. These data demonstrate that PM exposure induces DNA damage and activation of DNA damage response sensor MRN complex in macrophages. Disruption of MRN complex lead to persistent, unrepaired DNA damage that causes elevated inflammatory response.

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions

PubMed Central

Brem, Reto; Zhang, Xiaohui; Xu, Yao-Zhong; Karran, Peter

2015-01-01

Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C–I and C–Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion. PMID:25747491

Sexual rest and post-meiotic sperm ageing in house mice.

PubMed

Firman, R C; Young, F J; Rowe, D C; Duong, H T; Gasparini, C

2015-07-01

Fertilization by aged sperm can result in adverse fitness consequences for both males and females. Sperm storage during male sexual rest could provide an environment for post-meiotic sperm senescence causing a deterioration in the quality of stored sperm, possibly impacting on both sperm performance (e.g. swimming ability) and DNA quality. Here, we compared the proportion of sperm with fragmented DNA, an indicator of structural damage of DNA within the sperm cell, among males that had been sexually rested for approximately 2 months, to that of males that had mated recently. We found no evidence of intra-epididymal sperm DNA damage or any impairment in sperm performance, and consequently no evidence of post-meiotic sperm senescence. Our results suggest that male house mice are likely to possess mechanisms that function to ensure that their sperm reserves remain stocked with 'young', viable sperm during periods of sexual inactivity. We also discuss the possibility that our experimental design leads to no difference in the age of sperm among males from the two mating treatments. Post-meiotic sperm senescence is especially relevant under sperm competition. Thus, we sourced mice from populations that differed in their levels of post-copulatory sexual selection, enabling us to gain insight into how selection for higher sperm production influences the rate of sperm ageing and levels of DNA fragmentation. We found that males from the population that produced the highest number of sperm also had the smallest proportion of DNA-fragmented sperm and discuss this outcome in relation to selection acting upon males to ensure that they produce ejaculates with high-quality sperm that are successful in achieving fertilizations under competitive conditions. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

[Stress-induced cellular adaptive mutagenesis].

PubMed

Zhu, Linjiang; Li, Qi

2014-04-01

The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

Novel pyrimidinic selenourea induces DNA damage, cell cycle arrest, and apoptosis in human breast carcinoma.

PubMed

Barbosa, Flavio A R; Siminski, Tâmila; Canto, Rômulo F S; Almeida, Gabriela M; Mota, Nádia S R S; Ourique, Fabiana; Pedrosa, Rozangela Curi; Braga, Antonio Luiz

2018-06-11

Novel pyrimidinic selenoureas were synthesized and evaluated against tumour and normal cell lines. Among these, the compound named 3j initially showed relevant cytotoxicity and selectivity for tumour cells. Three analogues of 3j were designed and synthesized keeping in view the structural requirements of this compound. Almost all the tested compounds displayed considerable cytotoxicity. However, 8a, one of the 3j analogues, was shown to be highly selective and cytotoxic, especially for breast carcinoma cells (MCF-7) (IC 50  = 3.9 μM). Furthermore, 8a caused DNA damage, inhibited cell proliferation, was able to arrest cell cycle in S phase, and induced cell death by apoptosis in human breast carcinoma cells. Moreover, predictions of pharmacokinetic properties showed that 8a may present good absorption and permeation characteristics for oral administration. Overall, the current study established 8a as a potential drug prototype to be employed as a DNA interactive cytotoxic agent for the treatment of breast cancer. Copyright © 2018. Published by Elsevier Masson SAS.

DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Degui; Yu, Tianyu; Liu, Yongqiang

Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenicmore » mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. - Highlights: • This study explore contribution of DNA damage to neurodegeneration in Parkinson's disease mice. • A53T-α-Syn MEF cells show a prolonged DNA damage repair process and senescense phenotype. • DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice. • DNA damage decrease the number of nigrostriatal dopaminergic neurons. • Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages.« less

Understanding DNA under oxidative stress and sensitization: the role of molecular modeling

PubMed Central

Dumont, Elise; Monari, Antonio

2015-01-01

DNA is constantly exposed to damaging threats coming from oxidative stress, i.e., from the presence of free radicals and reactive oxygen species. Sensitization from exogenous and endogenous compounds that strongly enhance the frequency of light-induced lesions also plays an important role. The experimental determination of DNA lesions, though a difficult subject, is somehow well established and allows to elucidate even extremely rare DNA lesions. In parallel, molecular modeling has become fundamental to clearly understand the fine mechanisms related to DNA defects induction. Indeed, it offers an unprecedented possibility to get access to an atomistic or even electronic resolution. Ab initio molecular dynamics may also describe the time-evolution of the molecular system and its reactivity. Yet the modeling of DNA (photo-)reactions does necessitate elaborate multi-scale methodologies to tackle a damage induction reactivity that takes place in a complex environment. The double-stranded DNA environment is first characterized by a very high flexibility, but also a strongly inhomogeneous electrostatic embedding. Additionally, one aims at capturing more subtle effects, such as the sequence selectivity which is of critical important for DNA damage. The structure and dynamics of the DNA/sensitizers complexes, as well as the photo-induced electron- and energy-transfer phenomena taking place upon sensitization, should be carefully modeled. Finally the factors inducing different repair ratios for different lesions should also be rationalized. In this review we will critically analyze the different computational strategies used to model DNA lesions. A clear picture of the complex interplay between reactivity and structural factors will be sketched. The use of proper multi-scale modeling leads to the in-depth comprehension of DNA lesions mechanisms and also to the rational design of new chemo-therapeutic agents. PMID:26236706

Catch the live show: Visualizing damaged DNA in vivo.

PubMed

Oshidari, Roxanne; Mekhail, Karim

2018-06-01

The health of an organism is intimately linked to its ability to repair damaged DNA. Importantly, DNA repair processes are highly dynamic. This highlights the necessity of characterizing DNA repair in live cells. Advanced genome editing and imaging approaches allow us to visualize damaged DNA and its associated factors in real time. Here, we summarize both established and recent methods that are used to induce DNA damage and visualize damaged DNA and its repair in live cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Method for assaying clustered DNA damages

DOEpatents

Sutherland, Betsy M.

2004-09-07

Disclosed is a method for detecting and quantifying clustered damages in DNA. In this method, a first aliquot of the DNA to be tested for clustered damages with one or more lesion-specific cleaving reagents under conditions appropriate for cleavage of the DNA to produce single-strand nicks in the DNA at sites of damage lesions. The number average molecular length (Ln) of double stranded DNA is then quantitatively determined for the treated DNA. The number average molecular length (Ln) of double stranded DNA is also quantitatively determined for a second, untreated aliquot of the DNA. The frequency of clustered damages (.PHI..sub.c) in the DNA is then calculated.

Golden Berry-Derived 4β-hydroxywithanolide E for Selectively Killing Oral Cancer Cells by Generating ROS, DNA Damage, and Apoptotic Pathways

PubMed Central

Chiu, Chien-Chih; Haung, Jo-Wen; Chang, Fang-Rong; Huang, Kuang-Jing; Huang, Hsuan-Min; Huang, Hurng-Wern; Chou, Chon-Kit; Wu, Yang-Chang; Chang, Hsueh-Wei

2013-01-01

Background Most chemotherapeutic drugs for killing cancer cells are highly cytotoxic in normal cells, which limits their clinical applications. Therefore, a continuing challenge is identifying a drug that is hypersensitive to cancer cells but has minimal deleterious effects on healthy cells. The aims of this study were to evaluate the potential of 4β-hydroxywithanolide (4βHWE) for selectively killing cancer cells and to elucidate its related mechanisms. Methodology and Principal Findings Changes in survival, oxidative stress, DNA damage, and apoptosis signaling were compared between 4βHWE-treated oral cancer (Ca9-22) and normal fibroblast (HGF-1) cells. At 24 h and 48 h, the numbers of Ca9-22 cells were substantially decreased, but the numbers of HGF-1 cells were only slightly decreased. Additionally, the IC50 values for 4βHWE in the Ca9-22 cells were 3.6 and 1.9 µg/ml at 24 and 48 h, respectively. Time-dependent abnormal increases in ROS and dose-responsive mitochondrial depolarization can be exploited by using 4βHWE in chemotherapies for selectively killing cancer cells. Dose-dependent DNA damage measured by comet-nuclear extract assay and flow cytometry-based γ-H2AX/propidium iodide (PI) analysis showed relatively severer damage in the Ca9-22 cells. At both low and high concentrations, 4βHWE preferably perturbed the cell cycle in Ca9-22 cells by increasing the subG1 population and arrest of G1 or G2/M. Selective induction of apoptosis in Ca9-22 cells was further confirmed by Annexin V/PI assay, by preferential expression of phosphorylated ataxia-telangiectasia- and Rad3-related protein (p-ATR), and by cleavage of caspase 9, caspase 3, and poly ADP-ribose polymerase (PARP). Conclusions/Significance Together, the findings of this study, particularly the improved understanding of the selective killing mechanisms of 4βHWE, can be used to improve efficiency in killing oral cancer cells during chemoprevention and therapy. PMID:23705007

Aberrant DNA Damage Response Pathways May Predict the Outcome of Platinum Chemotherapy in Ovarian Cancer

PubMed Central

Stefanou, Dimitra T.; Bamias, Aristotelis; Episkopou, Hara; Kyrtopoulos, Soterios A.; Likka, Maria; Kalampokas, Theodore; Photiou, Stylianos; Gavalas, Nikos; Sfikakis, Petros P.; Dimopoulos, Meletios A.; Souliotis, Vassilis L.

2015-01-01

Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy. PMID:25659114

Lymphocyte DNA damage in Turkish asphalt workers detected by the comet assay.

PubMed

Bacaksiz, Aysegul; Kayaalti, Zeliha; Soylemez, Esma; Tutkun, Engin; Soylemezoglu, Tulin

2014-01-01

Asphalt has a highly complex structure and it contains several organic compounds including polycyclic aromatic hydrocarbons and heterocyclic compounds. In this study, comet assay was used to detect the DNA damage in blood lymphocytes of 30 workers exposed to asphalt fumes and 30 nonexposed controls. This is the first report on Turkish asphalt workers' investigated DNA damage using the alkaline single cell gel electrophoresis (SCGE). The DNA damage was evaluated by the percentage of DNA in the comet tail (% tail DNA) for each cell. According to our results, workers exposed to asphalt fumes had higher DNA damage than the control group (p < 0.01). The present study showed that asphalt fumes caused a significant increase in DNA damage and the comet assay is a suitable method for determining DNA damage in asphalt workers.

Assessment of antioxidative, chelating, and DNA-protective effects of selected essential oil components (eugenol, carvacrol, thymol, borneol, eucalyptol) of plants and intact Rosmarinus officinalis oil.

PubMed

Horvathova, Eva; Navarova, Jana; Galova, Eliska; Sevcovicova, Andrea; Chodakova, Lenka; Snahnicanova, Zuzana; Melusova, Martina; Kozics, Katarina; Slamenova, Darina

2014-07-16

Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe(2+)-induced damage. This paper evaluates the results in the light of experiences of other scientists.

Perspective on the pipeline of drugs being developed with modulation of DNA damage as a target.

PubMed

Plummer, Ruth

2010-09-15

Inhibitors of various elements of the DNA repair pathways have entered clinical development or are in late preclinical stages of drug development. It was initially considered that agents targeting DNA repair would act to overcome tumor resistance to chemotherapy and radiotherapy. More recent data have shown that targeting DNA repair pathways can be effective in selected tumors via a synthetically lethal route, with single agent activity having been shown with poly-ADP ribose polymerase (PARP) inhibitors. An increased understanding of the biology and interaction of the DNA repair pathways also means that rational combination of DNA repair inhibitors may also give great benefit in the clinic. ©2010 AACR.

DNA Damage and Repair: Relevance to Mechanisms of Neurodegeneration

PubMed Central

Martin, Lee J.

2008-01-01

DNA damage is a form of cell stress and injury that has been implicated in the pathogenesis of many neurologic disorders, including amyotrophic lateral sclerosis, Alzheimer disease, Down syndrome, Parkinson disease, cerebral ischemia, and head trauma. However, most data reveal only associations, and the role for DNA damage in direct mechanisms of neurodegeneration is vague with respect to being a definitive upstream cause of neuron cell death, rather than a consequence of the degeneration. Although neurons seem inclined to develop DNA damage during oxidative stress, most of the existing work on DNA damage and repair mechanisms has been done in the context of cancer biology using cycling non-neuronal cells but not nondividing (i.e. postmitotic) neurons. Nevertheless, the identification of mutations in genes that encode proteins that function in DNA repair and DNA damage response in human hereditary DNA repair deficiency syndromes and ataxic disorders is establishing a mechanistic precedent that clearly links DNA damage and DNA repair abnormalities with progressive neurodegeneration. This review summarizes DNA damage and repair mechanisms and their potential relevance to the evolution of degeneration in postmitotic neurons. PMID:18431258

Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation

PubMed Central

Zhang, Xurui; Ye, Caiyong; Sun, Fang; Wei, Wenjun; Hu, Burong; Wang, Jufang

2016-01-01

Persistent DNA damage is considered as a main cause of cellular senescence induced by ionizing radiation. However, the molecular bases of the DNA damage and their contribution to cellular senescence are not completely clear. In this study, we found that both heavy ions and X-rays induced senescence in human uveal melanoma 92–1 cells. By measuring senescence associated-β-galactosidase and cell proliferation, we identified that heavy ions were more effective at inducing senescence than X-rays. We observed less efficient repair when DNA damage was induced by heavy ions compared with X-rays and most of the irreparable damage was complex of single strand breaks and double strand breaks, while DNA damage induced by X-rays was mostly repaired in 24 hours and the remained damage was preferentially associated with telomeric DNA. Our results suggest that DNA damage induced by heavy ion is often complex and difficult to repair, thus presents as persistent DNA damage and pushes the cell into senescence. In contrast, persistent DNA damage induced by X-rays is preferentially associated with telomeric DNA and the telomere-favored persistent DNA damage contributes to X-rays induced cellular senescence. These findings provide new insight into the understanding of high relative biological effectiveness of heavy ions relevant to cancer therapy and space radiation research. PMID:27187621

Transcription and DNA Damage: Holding Hands or Crossing Swords?

PubMed

D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

2017-10-27

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mitochondrial DNA Damage and Diseases.

PubMed

Singh, Gyanesh; Pachouri, U C; Khaidem, Devika Chanu; Kundu, Aman; Chopra, Chirag; Singh, Pushplata

2015-01-01

Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA) damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments.

Interplay of space radiation and microgravity in DNA damage and DNA damage response.

PubMed

Moreno-Villanueva, María; Wong, Michael; Lu, Tao; Zhang, Ye; Wu, Honglu

2017-01-01

In space, multiple unique environmental factors, particularly microgravity and space radiation, pose constant threat to the DNA integrity of living organisms. Specifically, space radiation can cause damage to DNA directly, through the interaction of charged particles with the DNA molecules themselves, or indirectly through the production of free radicals. Although organisms have evolved strategies on Earth to confront such damage, space environmental conditions, especially microgravity, can impact DNA repair resulting in accumulation of severe DNA lesions. Ultimately these lesions, namely double strand breaks, chromosome aberrations, micronucleus formation, or mutations, can increase the risk for adverse health effects, such as cancer. How spaceflight factors affect DNA damage and the DNA damage response has been investigated since the early days of the human space program. Over the years, these experiments have been conducted either in space or using ground-based analogs. This review summarizes the evidence for DNA damage induction by space radiation and/or microgravity as well as spaceflight-related impacts on the DNA damage response. The review also discusses the conflicting results from studies aimed at addressing the question of potential synergies between microgravity and radiation with regard to DNA damage and cellular repair processes. We conclude that further experiments need to be performed in the true space environment in order to address this critical question.

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay.

PubMed

Kido, Ryoko; Sato, Itaru; Tsuda, Shuji

2006-01-01

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.

Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

PubMed Central

Cline, Susan D.

2012-01-01

How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

Sensing of dangerous DNA.

PubMed

Gasser, Stephan; Zhang, Wendy Y L; Tan, Nikki Yi Jie; Tripathi, Shubhita; Suter, Manuel A; Chew, Zhi Huan; Khatoo, Muznah; Ngeow, Joanne; Cheung, Florence S G

2017-07-01

The presence of damaged and microbial DNA can pose a threat to the survival of organisms. Cells express various sensors that recognize specific aspects of such potentially dangerous DNA. Recognition of damaged or microbial DNA by sensors induces cellular processes that are important for DNA repair and inflammation. Here, we review recent evidence that the cellular response to DNA damage and microbial DNA are tightly intertwined. We also discuss insights into the parameters that enable DNA sensors to distinguish damaged and microbial DNA from DNA present in healthy cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Track structure based modelling of light ion radiation effects on nuclear and mitochondrial DNA

NASA Astrophysics Data System (ADS)

Schmitt, Elke; Ottolenghi, Andrea; Dingfelder, Michael; Friedland, Werner; Kundrat, Pavel; Baiocco, Giorgio

2016-07-01

Space radiation risk assessment is of great importance for manned spaceflights in order to estimate risks and to develop counter-measures to reduce them. Biophysical simulations with PARTRAC can help greatly to improve the understanding of initial biological response to ionizing radiation. Results from modelling radiation quality dependent DNA damage and repair mechanisms up to chromosomal aberrations (e.g. dicentrics) can be used to predict radiation effects depending on the kind of mixed radiation field exposure. Especially dicentric yields can serve as a biomarker for an increased risk due to radiation and hence as an indicator for the effectiveness of the used shielding. PARTRAC [1] is a multi-scale biophysical research MC code for track structure based initial DNA damage and damage response modelling. It integrates physics, radiochemistry, detailed nuclear DNA structure and molecular biology of DNA repair by NHEJ-pathway to assess radiation effects on cellular level [2]. Ongoing experiments with quasi-homogeneously distributed compared to sub-micrometre focused bunches of protons, lithium and carbon ions allow a separation of effects due to DNA damage complexity on nanometre scale from damage clustering on (sub-) micrometre scale [3, 4]. These data provide an unprecedented benchmark for the DNA damage response model in PARTRAC and help understand the mechanisms leading to cell killing and chromosomal aberrations (e.g. dicentrics) induction. A large part of space radiation is due to a mixed ion field of high energy protons and few heavier ions that can be only partly absorbed by the shielding. Radiation damage induced by low-energy ions significantly contributes to the high relative biological efficiency (RBE) of ion beams around Bragg peak regions. For slow light ions the physical cross section data basis in PARTRAC has been extended to investigate radiation quality effects in the Bragg peak region [5]. The resulting range and LET values agree with ICRU data and SRIM calculations. Preliminary studies regarding the biological endpoints DSB (cluster) and chromosomal aberrations have been performed for selected light ions up to neon. Validation with experimental data as well as further calculations are underway and final results will be presented at the meeting. Mitochondrial alterations have been implicated in radiation-induced cardiovascular effects. To extend the applicability of PARTRAC biophysical tool towards effects on mitochondria, the nuclear DNA and chromatin as the primary target of radiation has been complemented by a model of mitochondrial DNA (mtDNA) to mimic a coronary cell with thousand mitochondria contained in the cytoplasm. Induced mtDNA damage (SSB, DSB) has been scored for 60Co photons and 5 MeV alpha-particle irradiation, assuming alternative radical scavenging capacities within the mitochondria. While direct radiation effects in mtDNA are identical to nuclear DNA, indirect effects in mtDNA are in general larger due to lower scavenging and the lack of DNA-protecting histones. These simulations complement the scarce experimental data on radiation-induced mtDNA damage and help elucidate the relative roles of initial mtDNA versus nuclear DNA damage and of pathways that amplify their respective effects. Ongoing and planned developments of PARTRAC include coupling with a radiation transport code and track-structure based calculations of cell killing for RBE studies on macroscopic scales within a mixed ion field. [1] Friedland, Dingfelder et al. (2011): "Track structures, DNA targets and radiation effects in the biophysical Monte Carlo simulation code PARTRAC", Mutat. Res. 711, 28-40 [2] Friedland et al. (2013): "Track structure based modelling of chromosome aberrations after photon and alpha-particle irradiation", Mutat. Res. 756, 213-223 [3] Schmid, Friedland et al. (2015): "Sub-micrometer 20 MeV protons or 45 MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks", Mutat. Res. 793, 30-40 [4] Friedland, Schmitt, Kundrat (2015): "Modelling Proton bunches focussed to submicrometre scales: Low-LET Radiation damage in high-LET-like spatial structure", Radiat. Prot. Dosim. 166, 34-37 [5] Schmitt, Friedland, Kundrat, Dingfelder, Ottolenghi (2015): "Cross section scaling for track structure simulations of low-energy ions in liquid water", Radiat. Prot. Dosim. 166, 15-18} Supported by the European Atomic Energy Community's Seventh Framework Programme (FP7/2007-2011) under grant agreement no 249689 "DoReMi" and the German Federal Ministry on Education and Research (KVSF-Projekt "LET-Verbund").

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

PubMed

Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

2017-11-22

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

The effects of metal ions on the DNA damage induced by hydrogen peroxide.

PubMed

Kobayashi, S; Ueda, K; Komano, T

1990-01-01

The effects of metal ions on DNA damage induced by hydrogen peroxide were investigated using two methods, agarose-gel electrophoretic analysis of supercoiled DNA and sequencing-gel analysis of single end-labeled DNA fragments of defined sequences. Hydrogen peroxide induced DNA damage when iron or copper ion was present. At least two classes of DNA damage were induced, one being direct DNA-strand cleavage, and the other being base modification labile to hot piperidine. The investigation of the damaged sites and the inhibitory effects of radical scavengers revealed that hydroxyl radical was the species which attacked DNA in the reaction of H2O2/Fe(II). On the other hand, two types of DNA damage were induced by H2O2/Cu(II). Type I damage was predominant and inhibited by potassium iodide, but type II was not. The sites of the base-modification induced by type I damage were similar to those by lipid peroxidation products and by ascorbate in the presence of Cu(II), suggesting the involvement of radical species other than free hydroxyl radical in the damaging reactions.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

PubMed Central

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F. Peter; Zhang, Huidong

2017-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

PubMed

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair.

PubMed

Khoronenkova, Svetlana V; Dianov, Grigory L

2013-02-01

The E3 ubiquitin ligase Mule/ARF-BP1 plays an important role in the cellular DNA damage response by controlling base excision repair and p53 protein levels. However, how the activity of Mule is regulated in response to DNA damage is currently unknown. Here, we report that the Ser18-containing isoform of the USP7 deubiquitylation enzyme (USP7S) controls Mule stability by preventing its self-ubiquitylation and subsequent proteasomal degradation. We find that in response to DNA damage, downregulation of USP7S leads to self-ubiquitylation and proteasomal degradation of Mule, which eventually leads to p53 accumulation. Cells that are unable to downregulate Mule show reduced ability to upregulate p53 levels in response to DNA damage. We also find that, as Mule inactivation is required for stabilization of base excision repair enzymes, the failure of cells to downregulate Mule after DNA damage results in deficient DNA repair. Our data describe a novel mechanism by which Mule is regulated in response to DNA damage and coordinates cellular DNA damage responses and DNA repair.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

PubMed

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PubMed

Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G(2)/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

Involvement of oxidatively damaged DNA and repair in cancer development and aging

PubMed Central

Tudek, Barbara; Winczura, Alicja; Janik, Justyna; Siomek, Agnieszka; Foksinski, Marek; Oliński, Ryszard

2010-01-01

DNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important factors in the development and pathology of an organism, including cancer. DNA is constantly damaged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) directly and also by products of lipid peroxidation (LPO), which form exocyclic adducts to DNA bases. A wide variety of oxidatively-generated DNA lesions are present in living cells. 8-oxoguanine (8-oxoGua) is one of the best known DNA lesions due to its mutagenic properties. Among LPO-derived DNA base modifications the most intensively studied are ethenoadenine and ethenocytosine, highly miscoding DNA lesions considered as markers of oxidative stress and promutagenic DNA damage. Although at present it is impossible to directly answer the question concerning involvement of oxidatively damaged DNA in cancer etiology, it is likely that oxidatively modified DNA bases may serve as a source of mutations that initiate carcinogenesis and are involved in aging (i.e. they may be causal factors responsible for these processes). To counteract the deleterious effect of oxidatively damaged DNA, all organisms have developed several DNA repair mechanisms. The efficiency of oxidatively damaged DNA repair was frequently found to be decreased in cancer patients. The present work reviews the basis for the biological significance of DNA damage, particularly effects of 8-oxoGua and ethenoadduct occurrence in DNA in the aspect of cancer development, drawing attention to the multiplicity of proteins with repair activities. PMID:20589166

Mitochondrial DNA Damage and Diseases

PubMed Central

Singh, Gyanesh; Pachouri, U C; Khaidem, Devika Chanu; Kundu, Aman; Chopra, Chirag; Singh, Pushplata

2015-01-01

Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA) damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments. PMID:27508052

DNA damage induced by ascorbate in the presence of Cu2+.

PubMed

Kobayashi, S; Ueda, K; Morita, J; Sakai, H; Komano, T

1988-01-25

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.

S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons.

PubMed

Ye, Weizhen; Blain, Stacy W

2010-08-01

A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimer's disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad3 related proteins, did not rescue apoptosis and in fact exacerbated death, suggesting that the DNA damage response might normally function neuroprotectively to block S phase-dependent apoptosis induction. As cell cycle events appear to be maintained in vivo in affected neurons for weeks to years before apoptosis is observed, activation of the DNA damage response might be able to hold cell cycle-induced death in check.

Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response.

PubMed

Ciccia, Alberto; Huang, Jen-Wei; Izhar, Lior; Sowa, Mathew E; Harper, J Wade; Elledge, Stephen J

2014-12-30

The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage.

Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response

PubMed Central

Ciccia, Alberto; Huang, Jen-Wei; Izhar, Lior; Sowa, Mathew E.; Harper, J. Wade; Elledge, Stephen J.

2014-01-01

The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage. PMID:25512513

PRP19 Transforms into a Sensor of RPA-ssDNA after DNA Damage and Drives ATR Activation via a Ubiquitin-Mediated Circuitry

PubMed Central

Maréchal, Alexandre; Wu, Ching-Shyi; Yazinski, Stephanie A.; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E.; Jin, Jianping; Zou, Lee

2014-01-01

Summary PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). While the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 binds RPA directly and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ATR kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, the recovery of stalled replication forks, and the progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. PMID:24332808

Oxidative damage to DNA: counterion-assisted addition of water to ionized DNA.

PubMed

Barnett, Robert N; Bongiorno, Angelo; Cleveland, Charles L; Joy, Abraham; Landman, Uzi; Schuster, Gary B

2006-08-23

Oxidative damage to DNA, implicated in mutagenesis, aging, and cancer, follows electron loss that generates a radical cation that migrates to a guanine, where it may react with water to form 8-oxo-7,8-dihydroguanine (8-OxoG). Molecular dynamics and ab initio quantum simulations on a B-DNA tetradecamer reveal activated reaction pathways that depend on the local counterion arrangement. The lowest activation barrier, 0.73 eV, is found for a reaction that starts from a configuration where a Na(+) resides in the major groove near the N7 atoms of adjacent guanines, and evolves through a transition state where a bond between a water oxygen atom and a carbon atom forms concurrently with displacement of a proton toward a neighboring water molecule. Subsequently, a bonded complex of a hydronium ion and the nearest backbone phosphate group forms. This counterion-assisted proton shuttle mechanism is supported by experiments exploiting selective substitution of backbone phosphates by methylphosphonates.

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours

PubMed Central

Xu, Hong; Di Antonio, Marco; McKinney, Steven; Mathew, Veena; Ho, Brandon; O'Neil, Nigel J.; Santos, Nancy Dos; Silvester, Jennifer; Wei, Vivien; Garcia, Jessica; Kabeer, Farhia; Lai, Daniel; Soriano, Priscilla; Banáth, Judit; Chiu, Derek S.; Yap, Damian; Le, Daniel D.; Ye, Frank B.; Zhang, Anni; Thu, Kelsie; Soong, John; Lin, Shu-chuan; Tsai, Angela Hsin Chin; Osako, Tomo; Algara, Teresa; Saunders, Darren N.; Wong, Jason; Xian, Jian; Bally, Marcel B.; Brenton, James D.; Brown, Grant W.; Shah, Sohrab P.; Cescon, David; Mak, Tak W.; Caldas, Carlos; Stirling, Peter C.; Hieter, Phil; Balasubramanian, Shankar; Aparicio, Samuel

2017-01-01

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016). PMID:28211448

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.

PubMed

Xu, Hong; Di Antonio, Marco; McKinney, Steven; Mathew, Veena; Ho, Brandon; O'Neil, Nigel J; Santos, Nancy Dos; Silvester, Jennifer; Wei, Vivien; Garcia, Jessica; Kabeer, Farhia; Lai, Daniel; Soriano, Priscilla; Banáth, Judit; Chiu, Derek S; Yap, Damian; Le, Daniel D; Ye, Frank B; Zhang, Anni; Thu, Kelsie; Soong, John; Lin, Shu-Chuan; Tsai, Angela Hsin Chin; Osako, Tomo; Algara, Teresa; Saunders, Darren N; Wong, Jason; Xian, Jian; Bally, Marcel B; Brenton, James D; Brown, Grant W; Shah, Sohrab P; Cescon, David; Mak, Tak W; Caldas, Carlos; Stirling, Peter C; Hieter, Phil; Balasubramanian, Shankar; Aparicio, Samuel

2017-02-17

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

Nanogravimetric and voltammetric DNA-hybridization biosensors for studies of DNA damage by common toxicants and pollutants.

PubMed

Nowicka, Anna M; Kowalczyk, Agata; Stojek, Zbigniew; Hepel, Maria

2010-01-01

Electrochemical and nanogravimetric DNA-hybridization biosensors have been developed for sensing single mismatches in the probe-target ssDNA sequences. The voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated tDNA. The mass-related frequency transduction was implemented by immobilizing the sensory pDNA on a gold-coated quartz crystal piezoresonators oscillating in the 10MHz band. The high sensitivity of these sensors enabled us to study DNA damage caused by representative toxicants and environmental pollutants, including Cr(VI) species, common pesticides and herbicides. We have found that the sensor responds rapidly to any damage caused by Cr(VI) species, with more severe DNA damage observed for Cr(2)O(7)(2-) and for CrO(4)(2-) in the presence of H(2)O(2) as compared to CrO(4)(2-) alone. All herbicides and pesticides examined caused DNA damage or structural alterations leading to the double-helix unwinding. Among these compounds, paraoxon-ethyl and atrazine caused the fastest and most severe damage to DNA. The physico-chemical mechanism of damaging interactions between toxicants and DNA has been proposed. The methodology of testing voltammetric and nanogravimetric DNA-hybridization biosensors developed in this work can be employed as a simple protocol to obtain rapid comparative data concerning DNA damage caused by herbicide, pesticides and other toxic pollutants. The DNA-hybridization biosensor can, therefore, be utilized as a rapid screening device for classifying environmental pollutants and to evaluate DNA damage induced by these compounds.

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

PubMed

Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

Investigation on the correlation between energy deposition and clustered DNA damage induced by low-energy electrons.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2018-05-01

This study presents the correlation between energy deposition and clustered DNA damage, based on a Monte Carlo simulation of the spectrum of direct DNA damage induced by low-energy electrons including the dissociative electron attachment. Clustered DNA damage is classified as simple and complex in terms of the combination of single-strand breaks (SSBs) or double-strand breaks (DSBs) and adjacent base damage (BD). The results show that the energy depositions associated with about 90% of total clustered DNA damage are below 150 eV. The simple clustered DNA damage, which is constituted of the combination of SSBs and adjacent BD, is dominant, accounting for 90% of all clustered DNA damage, and the spectra of the energy depositions correlating with them are similar for different primary energies. One type of simple clustered DNA damage is the combination of a SSB and 1-5 BD, which is denoted as SSB + BD. The average contribution of SSB + BD to total simple clustered DNA damage reaches up to about 84% for the considered primary energies. In all forms of SSB + BD, the SSB + BD including only one base damage is dominant (above 80%). In addition, for the considered primary energies, there is no obvious difference between the average energy depositions for a fixed complexity of SSB + BD determined by the number of base damage, but average energy depositions increase with the complexity of SSB + BD. In the complex clustered DNA damage constituted by the combination of DSBs and BD around them, a relatively simple type is a DSB combining adjacent BD, marked as DSB + BD, and it is of substantial contribution (on average up to about 82%). The spectrum of DSB + BD is given mainly by the DSB in combination with different numbers of base damage, from 1 to 5. For the considered primary energies, the DSB combined with only one base damage contributes about 83% of total DSB + BD, and the average energy deposition is about 106 eV. However, the energy deposition increases with the complexity of clustered DNA damage, and therefore, the clustered DNA damage with high complexity still needs to be considered in the study of radiation biological effects, in spite of their small contributions to all clustered DNA damage.

Somatic Rearrangement in B Cells: It's (Mostly) Nuclear Physics.

PubMed

Aiden, Erez Lieberman; Casellas, Rafael

2015-08-13

We discuss how principles of nuclear architecture drive typical gene rearrangements in B lymphocytes, whereas translocation hot spots and recurrent lesions reflect the extent of AID-mediated DNA damage and selection. Copyright © 2015 Elsevier Inc. All rights reserved.

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

PubMed Central

Pokrzywinski, Kaytee L.; Biel, Thomas G.; Kryndushkin, Dmitry; Rao, V. Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis. PMID:28030582

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

PubMed

Pokrzywinski, Kaytee L; Biel, Thomas G; Kryndushkin, Dmitry; Rao, V Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

Hereditary breast cancer and the BRCA1-associated FANCJ/BACH1/BRIP1

PubMed Central

Cantor, Sharon B; Guillemette, Shawna

2011-01-01

It is clear that FANCJ, also known as BACH1 or BRIP1, is an essential tumor suppressor gene based on the identification of clinically relevant mutations not only in breast cancer, but also the childhood cancer syndrome, Fanconi anemia. This conclusion is further supported by the direct and functional interaction between FANCJ and the hereditary breast cancer-associated gene product BRCA1. In the absence of the FANCJ DNA helicase or its interaction with BRCA1, cells have defects in several aspects of the DNA damage response. In particular, the BRCA1–FANCJ interaction is essential for promoting error-free repair, checkpoint control and for limiting DNA damage tolerance. As the number of FANCJ clinical mutations and affected patients accumulate, it will be critical to understand whether the associated tumors resemble BRCA-associated tumors. If so, FANCJ patients could also benefit from new therapies that selectively sensitize DNA repair-defective tumors and spare healthy cells. In this article, we summarize the breast cancer-associated FANCJ mutations and discuss functional outcomes for DNA repair and tumor suppression. PMID:21345144

Design checkpoint kinase 2 inhibitors by pharmacophore modeling and virtual screening techniques.

PubMed

Wang, Yen-Ling; Lin, Chun-Yuan; Shih, Kuei-Chung; Huang, Jui-Wen; Tang, Chuan-Yi

2013-12-01

Damage to DNA is caused by ionizing radiation, genotoxic chemicals or collapsed replication forks. When DNA is damaged or cells fail to respond, a mutation that is associated with breast or ovarian cancer may occur. Mammalian cells control and stabilize the genome using a cell cycle checkpoint to prevent damage to DNA or to repair damaged DNA. Checkpoint kinase 2 (Chk2) is one of the important kinases, which strongly affects DNA-damage and plays an important role in the response to the breakage of DNA double-strands and related lesions. Therefore, this study concerns Chk2. Its purpose is to find potential inhibitors using the pharmacophore hypotheses (PhModels) and virtual screening techniques. PhModels can identify inhibitors with high biological activities and virtual screening techniques are used to screen the database of the National Cancer Institute (NCI) to retrieve compounds that exhibit all of the pharmacophoric features of potential inhibitors with high interaction energy. Ten PhModels were generated using the HypoGen best algorithm. The established PhModel, Hypo01, was evaluated by performing a cost function analysis of its correlation coefficient (r), root mean square deviation (RMSD), cost difference, and configuration cost, with the values 0.955, 1.28, 192.51, and 16.07, respectively. The result of Fischer's cross-validation test for the Hypo01 model yielded a 95% confidence level, and the correlation coefficient of the testing set (rtest) had a best value of 0.81. The potential inhibitors were then chosen from the NCI database by Hypo01 model screening and molecular docking using the cdocker docking program. Finally, the selected compounds exhibited the identified pharmacophoric features and had a high interaction energy between the ligand and the receptor. Eighty-three potential inhibitors for Chk2 are retrieved for further study. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sperm DNA damage has a negative association with live-birth rates after IVF.

PubMed

Simon, L; Proutski, I; Stevenson, M; Jennings, D; McManus, J; Lutton, D; Lewis, S E M

2013-01-01

Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

NASA Astrophysics Data System (ADS)

Ramesh, Govindarajan; Wu, Honglu

Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

PubMed Central

Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

2016-01-01

Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

DNA damage, DNA susceptibility to oxidation and glutathione redox status in patients with Alzheimer's disease treated with and without memantine.

PubMed

Akkaya, Çağlayan; Yavuzer, Serap Sahin; Yavuzer, Hakan; Erkol, Gökhan; Bozluolcay, Melda; Dinçer, Yıldız

2017-07-15

The aim of the current study was to compare oxidative DNA damage, DNA susceptibility to oxidation, and ratio of GSH/GSSG in patients with Alzheimer's disease (AD) treated with acetylcholinesterase inhibitor (AChEI) and combined AChEI+memantine. The study included 67 patients with AD and 42 volunteers as control. DNA damage parameters (strand breaks, oxidized purines, H 2 O 2 -induced DNA damage) in lymphocyte DNA and GSH/GSSG ratio in erythrocytes were determined by the comet assay and spectrophotometric assay, respectively. DNA damage was found to be higher, GSH/GSSG ratio was found to be lower in the AD group than those in the control group. DNA strand breaks and H 2 O 2 -induced DNA damage were lower in the patients taking AChEI+memantine than those in the patients taking AChEI but no significant difference was determined between the groups for oxidized purines and GSH/GSSG ratio. In conclusion, increased systemic oxidative DNA damage and DNA susceptibility to oxidation may be resulted from diminished GSH/GSSG ratio in AD patients. Although DNA strand breaks and H 2 O 2 -induced DNA damage are lower in the AD patients treated with combined AChEI and memantine, this may not indicate protective effect of memantine against DNA oxidation due to similar levels of oxidized purines in the patients treated with AChEI and AChEI+memantine. Copyright © 2017 Elsevier B.V. All rights reserved.

Melittin induced cytogenetic damage, oxidative stress and changes in gene expression in human peripheral blood lymphocytes.

PubMed

Gajski, Goran; Domijan, Ana-Marija; Žegura, Bojana; Štern, Alja; Gerić, Marko; Novak Jovanović, Ivana; Vrhovac, Ivana; Madunić, Josip; Breljak, Davorka; Filipič, Metka; Garaj-Vrhovac, Vera

2016-02-01

Melittin (MEL) is the main constituent and principal toxin of bee venom. It is a small basic peptide, consisting of a known amino acid sequence, with powerful haemolytic activity. Since MEL is a nonspecific cytolytic peptide that attacks lipid membranes thus leading to toxicity, the presumption is that it could have significant therapeutic benefits. The aim was to evaluate the cyto/genotoxic effects of MEL in human peripheral blood lymphocytes (HPBLs) and the molecular mechanisms involved using a multi-biomarker approach. We found that MEL was cytotoxic for HPBLs in a dose- and time-dependent manner. It also induced morphological changes in the cell membrane, granulation and lysis of exposed cells. After treating HPBLs with non-cytotoxic concentrations of MEL, we observed increased DNA damage including oxidative DNA damage as well as increased formation of micronuclei and nuclear buds, and decreased lymphocyte proliferation determined by comet and micronucleus assays. The observed genotoxicity coincided with increased formation of reactive oxygen species, reduction of glutathione level, increased lipid peroxidation and phospholipase C activity, showing the induction of oxidative stress. MEL also modulated the expression of selected genes involved in DNA damage response (TP53, CDKN1A, GADD45α, MDM), oxidative stress (CAT, SOD1, GPX1, GSR and GCLC) and apoptosis (BAX, BCL-2, CAS-3 and CAS-7). Results indicate that MEL is genotoxic to HPBLs and provide evidence that oxidative stress is involved in its DNA damaging effects. MEL toxicity towards normal cells has to be considered if used for potential therapeutic purposes. Copyright © 2015 Elsevier Ltd. All rights reserved.

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

PubMed Central

Jangi, Mohini; Fleet, Christina; Cullen, Patrick; Gupta, Shipra V.; Mekhoubad, Shila; Chiao, Eric; Allaire, Norm; Bennett, C. Frank; Rigo, Frank; Krainer, Adrian R.; Hurt, Jessica A.; Carulli, John P.; Staropoli, John F.

2017-01-01

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death. PMID:28270613

Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.

PubMed

Guzder, S N; Sung, P; Prakash, L; Prakash, S

1998-11-20

Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

Visualizing the Search for Radiation-damaged DNA Bases in Real Time.

PubMed

Lee, Andrea J; Wallace, Susan S

2016-11-01

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

Visualizing the search for radiation-damaged DNA bases in real time

NASA Astrophysics Data System (ADS)

Lee, Andrea J.; Wallace, Susan S.

2016-11-01

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

DNA Damage in Euonymus japonicus Leaf Cells Caused by Roadside Pollution in Beijing

PubMed Central

Li, Tianxin; Zhang, Minjie; Gu, Ke; Herman, Uwizeyimana; Crittenden, John; Lu, Zhongming

2016-01-01

The inhalable particles from vehicle exhaust can cause DNA damage to exposed organisms. Research on DNA damage is primarily focused on the influence of specific pollutants on certain species or the effect of environmental pollution on human beings. To date, little research has quantitatively studied the relationship between roadside pollution and DNA damage. Based on an investigation of the roadside pollution in Beijing, Euonymus japonicus leaves of differing ages grown in heavily-polluted sections were chosen as biomonitors to detect DNA damage using the comet assay technique. The percentage of DNA in the tail and tail moment was chosen as the analysis index based on SPSS data analysis. The roadside samples showed significantly higher levels of DNA damage than non-roadside samples, which increased in older leaves, and the DNA damage to Euonymus japonicus leaf cells was positively correlated with haze-aggravated roadside pollution. The correlation between damage and the Air Quality Index (AQI) are 0.921 (one-year-old leaves), 0.894 (two-year-old leaves), and 0.878 (three-year-old leaves). Over time, the connection between DNA damage and AQI weakened, with the sensitivity coefficient for δyear 1 being larger than δyear 2 and δyear 3. These findings support the suitability and sensitivity of the comet assay for surveying plants for an estimation of DNA damage induced by environmental genotoxic agents. This study might be applied as a preliminary quantitative method for Chinese urban air pollution damage assessment caused by environmental stress. PMID:27455298

Lifestyle impacts on the aging associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

PubMed Central

Song, Zhangfa; von Figura, Guido; Liu, Yan; Kraus, Johann M.; Torrice, Chad; Dillon, Patric; Rudolph-Watabe, Masami; Ju, Zhenyu; Kestler, Hans A.; Sanoff, Hanna; Rudolph, K. Lenhard

2010-01-01

Summary Cellular aging is characterised by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and that activation of DNA damage responses. There is some evidence the DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging associated expression of serum markers of DNA damage (CRAMP, EF-1α, Stathmin, n-acetyl-glucosaminidase, and chitinase) in comparison to other described markers of cellular aging (p16INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age-independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age-independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16INK4a expression and negatively with telomere length in peripheral blood T-lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging. PMID:20560902

Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

PubMed

Xie, Ming-Zhang; Shoulkamy, Mahmoud I; Salem, Amir M H; Oba, Shunya; Goda, Mizuki; Nakano, Toshiaki; Ide, Hiroshi

2016-04-01

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of the CometChip platform to assess DNA damage in field-collected blood samples from turtles.

PubMed

Sykora, Peter; Chiari, Ylenia; Heaton, Andrew; Moreno, Nickolas; Glaberman, Scott; Sobol, Robert W

2018-05-01

DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

Selenium Potentiates Chemotherapeutic Selectivity: Improving Efficacy and Reducing Toxicity

DTIC Science & Technology

2007-04-01

regulates the rate-limiting step in global genomic repair through transcriptional control of the DNA damage recognition proteins xeroderma pigmentosum ...31). Xeroderma pigmentosum XPA cells defective in DNA repair served as a negative control for some experiments, as previously described (28). Cell...simian virus 40-transformed human cells. Mol Carcinog 2000;29:17–24. 14. Hwang BJ, Ford JM, Hanawalt PC, Chu G. Expression of the p48 xeroderma pigmentosum

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

PubMed

Sallustio, Benedetta C; Degraaf, Yvette C; Weekley, Josephine S; Burcham, Philip C

2006-05-01

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin > gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

PubMed

Wyatt, Lauren H; Luz, Anthony L; Cao, Xiou; Maurer, Laura L; Blawas, Ashley M; Aballay, Alejandro; Pan, William K Y; Meyer, Joel N

2017-04-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl 2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H 2 O 2 ), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl 2 , low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H 2 O 2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H 2 O 2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H 2 O 2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl 2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

PubMed Central

Wyatt, Lauren H.; Luz, Anthony L.; Cao, Xiou; Maurer, Laura L.; Blawas, Ashley M.; Aballay, Alejandro; Pan, William K.; Meyer, Joel N.

2017-01-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (~0.25 lesions/10 kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. PMID:28242054

Nanodosimetry of Low Energy (0.1 - 100 eV) Cation Damage to DNA

NASA Astrophysics Data System (ADS)

Sellami, L.; Martin, F.; Hunting, D.; Lacombe, S.; Huels, M. A.

2004-03-01

The importance of heavy ions in radiobiology is twofold: (1) they represent the most efficient and volume selective mode of radiotherapy of deep-seated and non-operable tumors, (2) in space environments, or at supersonic altitudes, the most lethal radiation consists of cosmic rays which have a high efficiency to induce clustered DNA lesions, mutations, and cancer. Thus, the study of their effects on DNA is essential for radiation risk assessment, dosimetry, and efficient use of hadrontherapy. Here, we investigate damage to DNA and its components, induced by heavy ion impact, via a novel ion-plasma method, which allows us to probe ion energy depositions in the 0.1-100 eV/nm range in nanoscopic biomolecular films. Cations are generated by electron impact in ultra pure gases (Ar, N2, CO, etc.), and are uniformly accelerated by grids towards the inside surface of a cylinder where an organic film was deposited. After ion irradiation at a specific energy and ion dose, the film is recovered and analyzed. For DNA, gel electrophoresis is used to quantify yields of single, double, and multiple strand breaks. For DNA components (mononucleotides), fragmentation and new products are measured by HPLC and MS.

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

PubMed Central

Schreiner, Sabrina; Nassal, Michael

2017-01-01

Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system. PMID:28531167

Genotoxin induced mutagenesis in the model plant Physcomitrella patens.

PubMed

Holá, Marcela; Kozák, Jaroslav; Vágnerová, Radka; Angelis, Karel J

2013-01-01

The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens

PubMed Central

Holá, Marcela; Kozák, Jaroslav; Vágnerová, Radka; Angelis, Karel J.

2013-01-01

The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype. PMID:24383055

Characterization of UVC-induced DNA damage in bloodstains: forensic implications.

PubMed

Hall, Ashley; Ballantyne, Jack

2004-09-01

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.

House dust mite-induced asthma causes oxidative damage and DNA double-strand breaks in the lungs.

PubMed

Chan, Tze Khee; Loh, Xin Yi; Peh, Hong Yong; Tan, W N Felicia; Tan, W S Daniel; Li, Na; Tay, Ian J J; Wong, W S Fred; Engelward, Bevin P

2016-07-01

Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) in vivo and in vitro. We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX) foci in bronchial epithelium. BEAS-2B cells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Machine learning classifier for identification of damaging missense mutations exclusive to human mitochondrial DNA-encoded polypeptides.

PubMed

Martín-Navarro, Antonio; Gaudioso-Simón, Andrés; Álvarez-Jarreta, Jorge; Montoya, Julio; Mayordomo, Elvira; Ruiz-Pesini, Eduardo

2017-03-07

Several methods have been developed to predict the pathogenicity of missense mutations but none has been specifically designed for classification of variants in mtDNA-encoded polypeptides. Moreover, there is not available curated dataset of neutral and damaging mtDNA missense variants to test the accuracy of predictors. Because mtDNA sequencing of patients suffering mitochondrial diseases is revealing many missense mutations, it is needed to prioritize candidate substitutions for further confirmation. Predictors can be useful as screening tools but their performance must be improved. We have developed a SVM classifier (Mitoclass.1) specific for mtDNA missense variants. Training and validation of the model was executed with 2,835 mtDNA damaging and neutral amino acid substitutions, previously curated by a set of rigorous pathogenicity criteria with high specificity. Each instance is described by a set of three attributes based on evolutionary conservation in Eukaryota of wildtype and mutant amino acids as well as coevolution and a novel evolutionary analysis of specific substitutions belonging to the same domain of mitochondrial polypeptides. Our classifier has performed better than other web-available tested predictors. We checked performance of three broadly used predictors with the total mutations of our curated dataset. PolyPhen-2 showed the best results for a screening proposal with a good sensitivity. Nevertheless, the number of false positive predictions was too high. Our method has an improved sensitivity and better specificity in relation to PolyPhen-2. We also publish predictions for the complete set of 24,201 possible missense variants in the 13 human mtDNA-encoded polypeptides. Mitoclass.1 allows a better selection of candidate damaging missense variants from mtDNA. A careful search of discriminatory attributes and a training step based on a curated dataset of amino acid substitutions belonging exclusively to human mtDNA genes allows an improved performance. Mitoclass.1 accuracy could be improved in the future when more mtDNA missense substitutions will be available for updating the attributes and retraining the model.

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

PubMed Central

Senevirathne, Mahinda; Kim, Soo-Hyun

2010-01-01

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro. PMID:20607062

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line.

PubMed

Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

2010-06-01

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

PubMed Central

Huang, Shar-yin N.; Murai, Junko; Dalla Rosa, Ilaria; Dexheimer, Thomas S.; Naumova, Alena; Gmeiner, William H.; Pommier, Yves

2013-01-01

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs. PMID:23775789

PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry.

PubMed

Maréchal, Alexandre; Li, Ju-Mei; Ji, Xiao Ye; Wu, Ching-Shyi; Yazinski, Stephanie A; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E; Jin, Jianping; Zou, Lee

2014-01-23

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA Damage and Repair in Human Cancer: Molecular Mechanisms and Contribution to Therapy-Related Leukemias

PubMed Central

Casorelli, Ida; Bossa, Cecilia; Bignami, Margherita

2012-01-01

Most antitumour therapies damage tumour cell DNA either directly or indirectly. Without repair, damage can result in genetic instability and eventually cancer. The strong association between the lack of DNA damage repair, mutations and cancer is dramatically demonstrated by a number of cancer-prone human syndromes, such as xeroderma pigmentosum, ataxia-telangiectasia and Fanconi anemia. Notably, DNA damage responses, and particularly DNA repair, influence the outcome of therapy. Because DNA repair normally excises lethal DNA lesions, it is intuitive that efficient repair will contribute to intrinsic drug resistance. Unexpectedly, a paradoxical relationship between DNA mismatch repair and drug sensitivity has been revealed by model studies in cell lines. This suggests that connections between DNA repair mechanism efficiency and tumour therapy might be more complex. Here, we review the evidence for the contribution of carcinogenic properties of several drugs as well as of alterations in specific mechanisms involved in drug-induced DNA damage response and repair in the pathogenesis of therapy-related cancers. PMID:23066388

Bio-monitoring of Tissue Accumulation and Genotoxic Effect of Heavy Metals in Cyprinus carpio from River Kabul Khyber Pakhtunkhwa Pakistan.

PubMed

Siraj, Muhammad; Khisroon, Muhammad; Khan, Ajmal; Zaidi, Farrah; Ullah, Ahmad; Rahman, Ghani

2018-03-01

The study explored (I) the concentration of heavy metals in water samples (II) their bioaccumulation in common carp Cyprinus carpio (III) and the subsequent genotoxicity in the selected organs of carp; from river Kabul, Khyber Pakhtunkhwa Pakistan. Except for Mercury (Hg) the water samples had all the heavy metals within permissible limits of recommended dietary allowance (RDA). Nonetheless a number of heavy metals (Zn, Ni, Cr, Cd, Pb and Hg) showed bioaccumulation at levels higher than permissible. Zinc (Zn) was the most while Cadmium (Cd) was the least accumulated metal in all tissue samples analyzed. The metal burden in different organs of C. carpio was in sequence of intestine > skin > liver > gills > muscle. The Comet assay established DNA damage in selected organs to be in accordance with metal burden; the most to least damage being in sequence of blood > intestine > skin > liver > gills > muscle. In conclusion assessment of DNA damage in the organs of C. carpio appears to be a useful bio-marker to evaluate genotoxic effects of heavy metal pollution.

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

NASA Technical Reports Server (NTRS)

Sutherland, Betsy M.; Georgakilas, Alexandros G.; Bennett, Paula V.; Laval, Jacques; Sutherland, John C.; Gewirtz, A. M. (Principal Investigator)

2003-01-01

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

PubMed Central

Hamperl, Stephan; Cimprich, Karlene A.

2014-01-01

Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

Direct Detection and Sequencing of Damaged DNA Bases

PubMed Central

2011-01-01

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

Direct detection and sequencing of damaged DNA bases.

PubMed

Clark, Tyson A; Spittle, Kristi E; Turner, Stephen W; Korlach, Jonas

2011-12-20

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

DNA damage and polyploidization.

PubMed

Chow, Jeremy; Poon, Randy Y C

2010-01-01

A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

PubMed

Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

2012-09-15

A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

Repair of Clustered Damage and DNA Polymerase Iota.

PubMed

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Noise Induced DNA Damage Within the Auditory Nerve.

PubMed

Guthrie, O'neil W

2017-03-01

An understanding of the molecular pathology that underlies noise induced neurotoxicity is a prerequisite to the design of targeted therapies. The objective of the current experiment was to determine whether or not DNA damage is part of the pathophysiologic sequela of noise induced neurotoxicity. The experiment consisted of 41 hooded Long-Evans rats (2 month old males) that were randomized into control and noise exposed groups. Both the control and the noise group followed the same time schedule and therefore started and ended the experiment together. The noise dose consisted of a 6000 Hz noise band at 105 dB SPL. Temporal bones from both groups were harvested, and immunohistochemistry was used to identify neurons with DNA damage. Quantitative morphometric analyses was then employed to determine the level of DNA damage. Neural action potentials were recorded to assess the functional impact of noise induced DNA damage. Immunohistochemical reactions revealed that the noise exposure precipitated DNA damage within the nucleus of auditory neurons. Quantitative morphometry confirmed the noise induced increase in DNA damage levels and the precipitation of DNA damage was associated with a significant loss of nerve sensitivity. Therefore, DNA damage is part of the molecular pathology that drives noise induced neurotoxicity. Anat Rec, 300:520-526, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

PubMed Central

Chen, Pin-I; Cao, Aiqin; Miyagawa, Kazuya; Tojais, Nancy F.; Hennigs, Jan K.; Li, Caiyun G.; Sweeney, Nathaly M.; Inglis, Audrey S.; Wang, Lingli; Li, Dan; Ye, Matthew; Feldman, Brian J.

2017-01-01

Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress. PMID:28138562

Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia.

PubMed

Aslan, Mehmet; Horoz, Mehmet; Kocyigit, Abdurrahim; Ozgonül, Saadet; Celik, Hakim; Celik, Metin; Erel, Ozcan

2006-10-10

Oxidant stress has been shown to play an important role in the pathogenesis of iron deficiency anemia. The aim of this study was to investigate the association between lymphocyte DNA damage, total antioxidant capacity and the degree of anemia in patients with iron deficiency anemia. Twenty-two female with iron deficiency anemia and 22 healthy females were enrolled in the study. Peripheral DNA damage was assessed using alkaline comet assay and plasma total antioxidant capacity was determined using an automated measurement method. Lymphocyte DNA damage of patients with iron deficiency anemia was significantly higher than controls (p<0.05), while total antioxidant capacity was significantly lower (p<0.001). While there was a positive correlation between total antioxidant capacity and hemoglobin levels (r=0.706, p<0.001), both total antioxidant capacity and hemoglobin levels were negatively correlated with DNA damage (r=-0.330, p<0.05 and r=-0.323, p<0.05, respectively). In conclusion, both oxidative stress and DNA damage are increased in IDA patients. Increased oxidative stress seems as an important factor that inducing DNA damage in those IDA patients. The relationships of oxidative stress and DNA damage with the severity of anemia suggest that both oxidative stress and DNA damage may, in part, have a role in the pathogenesis of IDA.

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

PubMed

Paul, Atanu; Wang, Bin

2017-05-18

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of basal DNA damage and oxidative stress in Wistar rat leukocytes after exposure to microwave radiation.

PubMed

Garaj-Vrhovac, Vera; Gajski, Goran; Trosić, Ivancica; Pavicić, Ivan

2009-05-17

The aim of this study was to assess whether microwave-induced DNA damage is basal or it is also generated through reactive oxygen species (ROS) formation. After having irradiated Wistar rats with 915MHz microwave radiation, we assessed different DNA alterations in peripheral leukocytes using standard and formamidopyrimidine DNA-glycosylase (Fpg)-modified comet assay. The first is a sensitive tool for detecting primary DNA damage, and the second is much more specific for detecting oxidative damage. The animals were irradiated for 1h a day for 2 weeks at a field power density of 2.4W/m(2), and the whole-body average specific absorption rate (SAR) of 0.6W/kg. Both the standard and the Fpg-modified comet assay detected increased DNA damage in blood leukocytes of the exposed rats. The significant increase in Fpg-detected DNA damage in the exposed rats suggests that oxidative stress is likely to be responsible. DNA damage detected by the standard comet assay indicates that some other mechanisms may also be involved. In addition, both methods served proved sensitive enough to measure basal and oxidative DNA damage after long-term exposure to 915MHz microwave radiation in vivo.

Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy

PubMed Central

Mishra, Manish; Lillvis, John; Seyoum, Berhane; Kowluru, Renu A.

2016-01-01

Purpose In the development of diabetic retinopathy, retinal mitochondria become dysfunctional, and mitochondrial DNA (mtDNA) is damaged. Because retinopathy is a progressive disease, and circulating glucose levels are high in diabetes, our aim was to investigate if peripheral blood mtDNA damage can serve as a potential biomarker of diabetic retinopathy. Methods Peripheral blood mtDNA damage was investigated by extended-length PCR in rats and mice, diabetic for 10 to 12 months (streptozotocin-induced, type 1 model), and in 12- and 40-week-old Zucker diabetic fatty rats (ZDF, type 2). Mitochondrial copy number (in gDNA) and transcription (in cDNA) were quantified by qPCR. Similar parameters were measured in blood from diabetic patients with/without retinopathy. Results Peripheral blood from diabetic rodents had significantly increased mtDNA damage and decreased copy numbers and transcription. Lipoic acid administration in diabetic rats, or Sod2 overexpression or MMP-9 knockdown in mice, the therapies that prevent diabetic retinopathy, also ameliorated blood mtDNA damage and restored copy numbers and transcription. Although blood from 40-week-old ZDF rats had significant mtDNA damage, 12-week-old rats had normal mtDNA. Diabetic patients with retinopathy had increased blood mtDNA damage, and decreased transcription and copy numbers compared with diabetic patients without retinopathy and nondiabetic individuals. Conclusions Type 1 diabetic rodents with oxidative stress modulated by pharmacologic/genetic means, and type 2 animal model and patients with/without diabetic retinopathy, demonstrate a strong relation between peripheral blood mtDNA damage and diabetic retinopathy, and suggest the possibility of use of peripheral blood mtDNA as a noninvasive biomarker of diabetic retinopathy. PMID:27494345

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

PubMed Central

Bhute, Vijesh J.; Palecek, Sean P.

2015-01-01

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage. PMID:26478723

Odorous Compounds from Poultry Manure Induce DNA Damage, Nuclear Changes, and Decrease Cell Membrane Integrity in Chicken Liver Hepatocellular Carcinoma Cells

PubMed Central

Matusiak, Katarzyna; Gałęcki, Remigiusz; Borowski, Sebastian; Gutarowska, Beata

2017-01-01

Animal breeding and management of organic wastes pose a serious problem to the health of livestock and workers, as well as the nearby residents. The aim of the present study was to determine the mechanisms of toxicity of selected common odorous compounds from poultry manure, including ammonia, dimethylamine (DMA), trimethylamine (TMA), butyric acid, phenol, and indole. We measured their genotoxic and cytotoxic activity in the model chicken cell line (LMH), in vitro, by comet assay and lactate dehydrogenase assay, respectively. We also made microscopic observations of any morphological changes in these cells by DAPI staining. Four compounds, namely ammonia, DMA, TMA, and butyric acid increased DNA damage in a dose-dependent manner (p < 0.05), reaching genotoxicity as high as 73.2 ± 1.9%. Phenol and indole induced extensive DNA damage independent of the concentration used. Ammonia, DMA, and TMA caused a dose-dependent release of lactate dehydrogenase (p < 0.05). The IC50 values were 0.02%, 0.05%, and 0.1% for DMA, ammonia and TMA, respectively. These compounds also induced nuclear morphological changes, such as chromatin condensation, shrinkage, nuclear fragmentation (apoptotic bodies), and chromatin lysis. Our study exhibited the damaging effects of odorous compounds in chick LMH cell line. PMID:28820500

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

PubMed Central

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

PubMed

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

CPTAC Develops Fit-for-Purpose Multiplex Immuno-MRM Assay for Profiling the DNA Damage Response Pathway | Office of Cancer Clinical Proteomics Research

Cancer.gov

Ionizing radiation (IR) is a commonly employed cancer treatment that kills cancer cells by damaging their DNA. While the DNA damage response (DDR) pathway may be key to determining tumor responses, radiochemical damage due to IR can target the patients’ healthy dividing cells, leading to the formation of secondary hematologic and solid tumors after DNA-damaging therapy.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

PubMed Central

Ding, Wei; Bishop, Michelle E.; Lyn-Cook, Lascelles E.; Davis, Kelly J.; Manjanatha, Mugimane G.

2016-01-01

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals. PMID:27166647

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.

PubMed

Ding, Wei; Bishop, Michelle E; Lyn-Cook, Lascelles E; Davis, Kelly J; Manjanatha, Mugimane G

2016-05-04

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

Colorimetric detection of DNA damage by using hemin-graphene nanocomposites

NASA Astrophysics Data System (ADS)

Wei, W.; Zhang, D. M.; Yin, L. H.; Pu, Y. P.; Liu, S. Q.

2013-04-01

A colorimetric method for detection of DNA damage was developed by using hemin-graphene nanosheets (H-GNs). H-GNs were skillfully synthesized by adsorping of hemin on graphene through π-π interactions. The as-prepared H-GNs possessed both the ability of graphene to differentiate the damage DNA from intact DNA and the catalytic action of hemin. The damaged DNA made H-GNs coagulated to different degrees from the intact DNA because there were different amount of negative charge exposed on their surface, which made a great impact on the solubility of H-GNs. As a result, the corresponding centrifugal supernatant of H-GNs solution showed different color in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, which could be discriminated by naked eyes or by ultraviolet (UV)-visible spectrometer. Based on this, the damaged effects of styrene oxide (SO), NaAsO2 and UV radiation on DNA were studied. Results showed that SO exerted most serious damage effect on DNA although all of them damaged DNA seriously. The new method for detection of DNA damage showed good prospect in the evaluation of genotoxicity of new compounds, the maximum limit of pesticide residue, food additives, and so on, which is important in the fields of food science, pharmaceutical science and pesticide science.

DNA Damage, DNA Repair, Aging, and Neurodegeneration

PubMed Central

Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.

2015-01-01

Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

Oxidative Stress Mechanisms Do Not Discriminate between Genotoxic and Nongenotoxic Liver Carcinogens.

PubMed

Deferme, Lize; Wolters, Jarno; Claessen, Sandra; Briedé, Jacco; Kleinjans, Jos

2015-08-17

It is widely accepted that in chemical carcinogenesis different modes-of-action exist, e.g., genotoxic (GTX) versus nongenotoxic (NGTX) carcinogenesis. In this context, it has been suggested that oxidative stress response pathways are typical for NGTX carcinogenesis. To evaluate this, we examined oxidative stress-related changes in gene expression, cell cycle distribution, and (oxidative) DNA damage in human hepatoma cells (HepG2) exposed to GTX-, NGTX-, and noncarcinogens, at multiple time points (4-8-24-48-72 h). Two GTX (azathriopine (AZA) and furan) and two NGTX (tetradecanoyl-phorbol-acetate, (TPA) and tetrachloroethylene (TCE)) carcinogens as well as two noncarcinogens (diazinon (DZN, d-mannitol (Dman)) were selected, while per class one compound was deemed to induce oxidative stress and the other not. Oxidative stressors AZA, TPA, and DZN induced a 10-fold higher number of gene expression changes over time compared to those of furan, TCE, or Dman treatment. Genes commonly expressed among AZA, TPA, and DZN were specifically involved in oxidative stress, DNA damage, and immune responses. However, differences in gene expression between GTX and NGTX carcinogens did not correlate to oxidative stress or DNA damage but could instead be assigned to compound-specific characteristics. This conclusion was underlined by results from functional readouts on ROS formation and (oxidative) DNA damage. Therefore, oxidative stress may represent the underlying cause for increased risk of liver toxicity and even carcinogenesis; however, it does not discriminate between GTX and NGTX carcinogens.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA damage in an animal model of maple syrup urine disease.

PubMed

Scaini, Giselli; Jeremias, Isabela C; Morais, Meline O S; Borges, Gabriela D; Munhoz, Bruna P; Leffa, Daniela D; Andrade, Vanessa M; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2012-06-01

Maple syrup urine disease is an inborn error of metabolism caused by a severe deficiency of the branched chain alpha-ketoacid dehydrogenase complex. Neurological dysfunction is a common finding in patients with maple syrup urine disease. However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly understood. In this study, we investigated whether acute or chronic administration of a branched chain amino acid pool (leucine, isoleucine and valine) causes transient DNA damage, as determined by the alkaline comet assay, in the brain and blood of rats during development and whether antioxidant treatment prevented the alterations induced by branched chain amino acids. Our results showed that the acute administration of branched chain amino acids increased the DNA damage frequency and damage index in the hippocampus. However, the chronic administration of branched chain amino acids increased the DNA damage frequency and damage index in both the hippocampus and the striatum, and the antioxidant treatment was able to prevent DNA damage in the hippocampus and striatum. The present study demonstrated that metabolite accumulation in MSUD induces DNA damage in the hippocampus and striatum and that it may be implicated in the neuropathology observed in the affected patients. We demonstrated that the effect of antioxidant treatment (N-acetylcysteine plus deferoxamine) prevented DNA damage, suggesting the involvement of oxidative stress in DNA damage. Copyright © 2012 Elsevier Inc. All rights reserved.

Melanoma prevention by MC1R selective small peptide analogs of alpha MSH | Division of Cancer Prevention

Cancer.gov

This revised application will test the hypothesis that small peptide analogs of ¿-melanocortin (¿-MSH) that are selective agonists of the melanocortin 1 receptor (MC1R) will prevent melanoma tumor formation in transgenic mouse melanoma models by enhancing repair of ultraviolet radiation (UV)-induced DNA damage and stimulating melanogenesis. We have pioneered the research on

Clinical Study of Ursodeoxycholic Acid in Barrett’s Esophagus Patients

PubMed Central

Banerjee, Bhaskar; Shaheen, Nicholas J.; Martinez, Jessica A.; Hsu, Chiu-Hsieh; Trowers, Eugene; Gibson, Blake A.; Della’Zanna, Gary; Richmond, Ellen; Chow, H-H. Sherry

2016-01-01

Prior research strongly implicates gastric acid and bile acids, two major components of the gastroesophageal refluxate, in the development of Barrett’s esophagus (BE) and its pathogenesis. Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has been shown to protect esophageal cells against oxidative stress induced by cytotoxic bile acids. We conducted a pilot clinical study to evaluate the clinical activity of UDCA in patients with BE. Twenty-nine BE patients received UDCA treatment at a daily dose of 13–15 mg/kg/day for six months. The clinical activity of UDCA was assessed by evaluating changes in gastric bile acid composition and markers of oxidative DNA damage (8-hydroxydeoxyguanosine, 8OHdG), cell proliferation (Ki67), and apoptosis (cleaved caspase 3, CC3) in BE epithelium. The bile acid concentrations in gastric fluid were measured by liquid chromatography-mass spectrometry. At baseline, UDCA (sum of unchanged and glycine/taurine conjugates) accounted for 18.2% of total gastric bile acids. Post UDCA intervention, UDCA increased significantly to account for 93.39% of total gastric bile acids (p<0.0001). The expression of markers of oxidative DNA damage, cell proliferation, and apoptosis was assessed in the BE biopsies by immunohistochemistry. The selected tissue biomarkers were unchanged after 6 months of UDCA intervention. We conclude that high dose UDCA supplementation for six months resulted in favorable changes in gastric bile acid composition but did not modulate selected markers of oxidative DNA damage, cell proliferation, and apoptosis in the BE epithelium. PMID:26908564

Modulation of inflammation and disease tolerance by DNA damage response pathways.

PubMed

Neves-Costa, Ana; Moita, Luis F

2017-03-01

The accurate replication and repair of DNA is central to organismal survival. This process is challenged by the many factors that can change genetic information such as replication errors and direct damage to the DNA molecule by chemical and physical agents. DNA damage can also result from microorganism invasion as an integral step of their life cycle or as collateral damage from host defense mechanisms against pathogens. Here we review the complex crosstalk of DNA damage response and immune response pathways that might be evolutionarily connected and argue that DNA damage response pathways can be explored therapeutically to induce disease tolerance through the activation of tissue damage control processes. Such approach may constitute the missing pillar in the treatment of critical illnesses caused by multiple organ failure, such as sepsis and septic shock. © 2016 Federation of European Biochemical Societies.

Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation.

PubMed

Tokuyama, Yuka; Furusawa, Yoshiya; Ide, Hiroshi; Yasui, Akira; Terato, Hiroaki

2015-05-01

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm(-1), respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay.

PubMed

Seidel, Clemens; Lautenschläger, Christine; Dunst, Jürgen; Müller, Arndt-Christian

2012-04-20

To investigate whether different conditions of DNA structure and radiation treatment could modify heterogeneity of response. Additionally to study variance as a potential parameter of heterogeneity for radiosensitivity testing. Two-hundred leukocytes per sample of healthy donors were split into four groups. I: Intact chromatin structure; II: Nucleoids of histone-depleted DNA; III: Nucleoids of histone-depleted DNA with 90 mM DMSO as antioxidant. Response to single (I-III) and twice (IV) irradiation with 4 Gy and repair kinetics were evaluated using %Tail-DNA. Heterogeneity of DNA damage was determined by calculation of variance of DNA-damage (V) and mean variance (Mvar), mutual comparisons were done by one-way analysis of variance (ANOVA). Heterogeneity of initial DNA-damage (I, 0 min repair) increased without histones (II). Absence of histones was balanced by addition of antioxidants (III). Repair reduced heterogeneity of all samples (with and without irradiation). However double irradiation plus repair led to a higher level of heterogeneity distinguishable from single irradiation and repair in intact cells. Increase of mean DNA damage was associated with a similarly elevated variance of DNA damage (r = +0.88). Heterogeneity of DNA-damage can be modified by histone level, antioxidant concentration, repair and radiation dose and was positively correlated with DNA damage. Experimental conditions might be optimized by reducing scatter of comet assay data by repair and antioxidants, potentially allowing better discrimination of small differences. Amount of heterogeneity measured by variance might be an additional useful parameter to characterize radiosensitivity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srinivas, L.; Shalini, V.K.

Twigs-dry leaves smoke condensate (TDS), as a source of clastogenic ROS and carcinogenic PAH, was investigated for its in vitro DNA-damaging effect in calf thymus DNA and human peripheral lymphocytes. An aqueous turmeric component--Aq.T--with an established antioxidant activity, was tested as a DNA protectant. TDS induced 13-fold damage to calf thymus DNA as judged by the emergence of a DNA damage specific, fluorescent product (em: 405 nm). Aq.T at 800 ng/microL extended 69% protection to calf thymus DNA and was comparable to the other protectants such as curcumin, BHA, vitamin E, SOD, and CAT. In human peripheral lymphocytes, TDS inducedmore » extensive DNA damage in comparison with the tumor promoter TPA, as judged by FADU. Aq.T at 300 ng/microL extended 90% protection to human lymphocyte DNA against TDS-induced damage, and was more effective than the other protectants--DABCO, D-mannitol, sodium benzoate, vitamin E (ROS quenchers), SOD, CAT (antioxidant enzymes), tannic acid, flufenamic acid, BHA, BHT, n-PG, curcumin and quercetin (antioxidants). Aq.T offered 65% protection to human lymphocyte DNA against TPA-induced damage and was comparable to SOD. The above results indicate that TDS induces substantial DNA damage in calf thymus DNA and human lymphocytes and Aq.T is an efficient protectant.« less

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Preterm newborns show slower repair of oxidative damage and paternal smoking associated DNA damage.

PubMed

Vande Loock, Kim; Ciardelli, Roberta; Decordier, Ilse; Plas, Gina; Haumont, Dominique; Kirsch-Volders, Micheline

2012-09-01

Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero, increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H(2)O(2) induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

Phosphorylation of human INO80 is involved in DNA damage tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kato, Dai; Waki, Mayumi; Umezawa, Masaki

Highlights: Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced PCNA ubiquitination. Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. Black-Right-Pointing-Pointer Western blot analyses showed phosphorylated hINO80 C-terminus. Black-Right-Pointing-Pointer Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in themore » DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.« less

Recent Advances in the Structural Mechanisms of DNA Glycosylases

PubMed Central

Brooks, Sonja C.; Adhikary, Suraj; Rubinson, Emily H.; Eichman, Brandt F.

2012-01-01

DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28 years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities. PMID:23076011

DNA damage in cells exhibiting radiation-induced genomic instability

DOE PAGES

Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

2015-02-22

Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesismore » that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.« less

Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

NASA Technical Reports Server (NTRS)

Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

2000-01-01

Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

A novel transcription factor gene FHS1 is involved in the DNA damage response in Fusarium graminearum

PubMed Central

Son, Hokyoung; Fu, Minmin; Lee, Yoonji; Lim, Jae Yun; Min, Kyunghun; Kim, Jin-Cheol; Choi, Gyung Ja; Lee, Yin-Won

2016-01-01

Cell cycle regulation and the maintenance of genome integrity are crucial for the development and virulence of the pathogenic plant fungus Fusarium graminearum. To identify transcription factors (TFs) related to these processes, four DNA-damaging agents were applied to screen a F. graminearum TF mutant library. Sixteen TFs were identified to be likely involved in DNA damage responses. Fhs1 is a fungal specific Zn(II)2Cys6 TF that localises exclusively to nuclei. fhs1 deletion mutants were hypersensitive to hydroxyurea and defective in mitotic cell division. Moreover, deletion of FHS1 resulted in defects in perithecia production and virulence and led to the accumulation of DNA damage. Our genetic evidence demonstrated that the FHS1-associated signalling pathway for DNA damage response is independent of the ATM or ATR pathways. This study identified sixteen genes involved in the DNA damage response and is the first to characterise the novel transcription factor gene FHS1, which is involved in the DNA damage response. The results provide new insights into mechanisms underlying DNA damage responses in fungi, including F. graminearum. PMID:26888604

Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells.

PubMed

Bru, Samuel; Samper-Martín, Bàrbara; Quandt, Eva; Hernández-Ortega, Sara; Martínez-Laínez, Joan M; Garí, Eloi; Rafel, Marta; Torres-Torronteras, Javier; Martí, Ramón; Ribeiro, Mariana P C; Jiménez, Javier; Clotet, Josep

2017-09-01

Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

PubMed

Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

2016-11-29

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

PubMed Central

Hill, Sarah J.; Mordes, Daniel A.; Cameron, Lisa A.; Neuberg, Donna S.; Landini, Serena; Eggan, Kevin; Livingston, David M.

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis. PMID:27849576

In vitro non-homologous DNA end joining assays—The 20th anniversary

PubMed Central

Pastwa, Elzbieta; Somiari, Richard I.; Malinowski, Mariusz; Somiari, Stella B.; Winters, Thomas A.

2010-01-01

DNA double-strand breaks (DSBs) are the most serious forms of DNA damage in cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to mutations or cell death, and consequently, to cancer predisposition. In human cells non-homologous DNA end joining (NHEJ) is the main repair mechanism of these breaks. Systems for DNA end joining study have been developing during the last 20 years. New assays have some advantages over earlier in vitro DSBs repair assays because they are less time-consuming, allow the use of clinical material and examination of the joining DNA ends produced physiologically in mammalian cells. Proteins involved in NHEJ repair pathway can serve as biomarkers or molecular targets for anticancer drugs. Results of studies on NHEJ in cancer could help to select potent repair inhibitors that may selectively sensitize tumor cells to ionizing radiation (IR) and chemotherapy. Here, we review the principles and practice of in vitro NHEJ assays and provide some insights into the future prospects of this assay in cancer diagnosis and treatment. PMID:19110069

DNA damage in blood cells in relation to chemotherapy and nutritional status in colorectal cancer patients-A pilot study.

PubMed

Kværner, Ane Sørlie; Minaguchi, Jun; Yamani, Naouale El; Henriksen, Christine; Ræder, Hanna; Paur, Ingvild; Henriksen, Hege Berg; Wiedswang, Gro; Smeland, Sigbjørn; Blomhoff, Rune; Collins, Andrew Richard; Bøhn, Siv Kjølsrud

2018-03-01

DNA damage can be considered as a biomarker for toxicity and response to chemotherapy. It is not known whether the chemotherapy-induced genotoxicity is associated with malnutrition. In this pilot study, we assess genotoxicity by means of DNA damage in patients with lymph-node positive colorectal cancer (CRC) and explore associations with chemotherapy treatment and nutritional status. DNA damage was compared between patients receiving chemotherapy (n = 24) and those not receiving chemotherapy (n = 20). DNA damage was measured in frozen whole blood by the comet assay. Associations between DNA damage and various indicators of malnutrition were also explored, including Patient-Generated Subjective Global Assessment (PG-SGA), bioelectrical impedance analysis (BIA) and anthropometric measurements, using multiple linear regression models. Patients on chemotherapy have higher levels of DNA damage in blood cells than patients not receiving chemotherapy (median of 16.9 and 7.9% tail DNA respectively, p = 0.001). The moderately malnourished patients (PG-SGA category B), representing 41% of the patients, have higher levels of cellular DNA damage than patients with good nutritional status (mean difference of 7.5% tail DNA, p = 0.033). In conclusion, adjuvant chemotherapy and malnutrition are both associated with increased levels of DNA damage in blood cells of CRC patients. Carefully controlled longitudinal studies or randomized controlled trials should be performed to determine whether good nutritional status may protect against chemotherapy-induced genotoxicity and enhance compliance to therapy in CRC patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Radioprotective effects of honeybee venom (Apis mellifera) against 915-MHz microwave radiation-induced DNA damage in wistar rat lymphocytes: in vitro study.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2009-01-01

The aim of this study is to investigate the radioprotective effect of bee venom against DNA damage induced by 915-MHz microwave radiation (specific absorption rate of 0.6 W/kg) in Wistar rats. Whole blood lymphocytes of Wistar rats are treated with 1 microg/mL bee venom 4 hours prior to and immediately before irradiation. Standard and formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assays are used to assess basal and oxidative DNA damage produced by reactive oxygen species. Bee venom shows a decrease in DNA damage compared with irradiated samples. Parameters of Fpg-modified comet assay are statistically different from controls, making this assay more sensitive and suggesting that oxidative stress is a possible mechanism of DNA damage induction. Bee venom is demonstrated to have a radioprotective effect against basal and oxidative DNA damage. Furthermore, bee venom is not genotoxic and does not produce oxidative damage in the low concentrations used in this study.

Unrepaired clustered DNA lesions induce chromosome breakage in human cells

PubMed Central

Asaithamby, Aroumougame; Hu, Burong; Chen, David J.

2011-01-01

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis. PMID:21527720

Enterococcus faecalis infection causes inflammation, intracellular oxphos-independent ROS production, and DNA damage in human gastric cancer cells.

PubMed

Strickertsson, Jesper A B; Desler, Claus; Martin-Bertelsen, Tomas; Machado, Ana Manuel Dantas; Wadstrøm, Torkel; Winther, Ole; Rasmussen, Lene Juel; Friis-Hansen, Lennart

2013-01-01

Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-κB inflammatory response as well as impaired DNA damage response and cell cycle control gene expression. Array Express accession number E-MEXP-3496.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarwar, Tarique; Zafaryab, Md; Husain, Mohammed Amir

Ferulic acid (FA) is a plant polyphenol showing diverse therapeutic effects against cancer, diabetes, cardiovascular and neurodegenerative diseases. FA is a known antioxidant at lower concentrations, however at higher concentrations or in the presence of metal ions such as copper, it may act as a pro-oxidant. It has been reported that copper levels are significantly raised in different malignancies. Cancer cells are under increased oxidative stress as compared to normal cells. Certain therapeutic substances like polyphenols can further increase this oxidative stress and kill cancer cells without affecting the proliferation of normal cells. Through various in vitro experiments we havemore » shown that the pro-oxidant properties of FA are enhanced in the presence of copper. Comet assay demonstrated the ability of FA to cause oxidative DNA breakage in human peripheral lymphocytes which was ameliorated by specific copper-chelating agent such as neocuproine and scavengers of ROS. This suggested the mobilization of endogenous copper in ROS generation and consequent DNA damage. These results were further validated through cytotoxicity experiments involving different cell lines. Thus, we conclude that such a pro-oxidant mechanism involving endogenous copper better explains the anticancer activities of FA. This would be an alternate non-enzymatic, and copper-mediated pathway for the cytotoxic activities of FA where it can selectively target cancer cells with elevated levels of copper and ROS. - Highlights: • Pro-oxidant properties of ferulic acid are enhanced in presence of copper. • Ferulic acid causes oxidative DNA damage in lymphocytes as observed by comet assay. • DNA damage was ameliorated by copper chelating agent neocuproine and ROS scavengers. • Endogenous copper is involved in ROS generation causing DNA damage. • Ferulic acid exerts cancer cell specific cytotoxicity as observed by MTT assay.« less

DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

NASA Astrophysics Data System (ADS)

Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

2015-04-01

The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

Engineering a Cell-surface Aptamer Circuit for Targeted and Amplified Photodynamic Cancer Therapy

PubMed Central

Han, Da; Zhu, Guizhi; Wu, Cuichen; Zhu, Zhi; Chen, Tao; Zhang, Xiaobing

2013-01-01

Photodynamic therapy (PDT) is one of the most promising and noninvasive methods for clinical treatment of different malignant diseases. Here, we present a novel strategy of designing an aptamer-based DNA nanocircuit capable of the selective recognition of cancer cells, controllable activation of photosensitizer and amplification of photodynamic therapeutic effect. The aptamers can selectively recognize target cancer cells and bind to the specific proteins on cell membranes. Then the overhanging catalyst sequence on aptamer can trigger a toehold-mediated catalytic strand displacement to activate photosensitizer and achieve amplified therapeutic effect. The specific binding-induced activation allows the DNA circuit to distinguish diseased cells from healthy cells, reducing damage to nearby healthy cells. Moreover, the catalytic amplification reaction will only take place close to the target cancer cells, resulting in a high local concentration of singlet oxygen to selectively kill the target cells. The principle employed in this study demonstrated the feasibility of assembling a DNA circuit on cell membranes and could further broaden the utility of DNA circuits for applications in biology, biotechnology, and biomedicine. PMID:23397942

Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage.

PubMed

Perron, Nathan R; García, Carla R; Pinzón, Julio R; Chaur, Manuel N; Brumaghim, Julia L

2011-05-01

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC(50)) for most of the tested polyphenols are between 8 and 480 μM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu(I)-mediated DNA damage less effectively than Fe(II)-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu(I), and H(2)O(2), it is likely that weak polyphenol-Cu(I) interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu(I), H(2)O(2), and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy. Copyright © 2011 Elsevier Inc. All rights reserved.

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis[OPEN

PubMed Central

Liu, Chun-Hsin; Finke, Andreas; Díaz, Mariana; Rozhon, Wilfried; Poppenberger, Brigitte; Baubec, Tuncay; Pecinka, Ales

2015-01-01

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway. PMID:26023162

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

PubMed Central

Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

2016-01-01

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

Mathematical Methods for Studying DNA and Protein Interactions

NASA Astrophysics Data System (ADS)

LeGresley, Sarah

Deoxyribnucleic Acid (DNA) damage can lead to health related issues such as developmental disorders, aging, and cancer. It has been estimated that damage rates may be as high as 100,000 per cell per day. Because of the devastating effects that DNA damage can have, DNA repair mechanisms are of great interest yet are not completely understood. To gain a better understanding of possible DNA repair mechanisms, my dissertation focused on mathematical methods for understanding the interactions between DNA and proteins. I developed a damaged DNA model to estimate the probabilities of damaged DNA being located at specific positions. Experiments were then performed that suggested that the damaged DNA may be repositioned. These experimental results were consistent with the model's prediction that damaged DNA has preferred locations. To study how proteins might be moving along the DNA, I studied the use of the uniform motion "n-step" model. The n-step model has been used to determine the kinetics parameters (e.g. rates at which a protein moves along the DNA, how much energy is required to move a protein along a specified amount of DNA, etc.) of proteins moving along the DNA. Monte Carlo methods were used to simulate proteins moving with different types of non-uniform motion (e.g. backward, jumping, etc.) along the DNA. Estimates for the kinetics parameters in the n-step model were found by fitting of the Monte Carlo simulation data. Analysis indicated that non-uniform motion of the protein may lead to over or underestimation of the kinetic parameters of this n-step model.

Biorecognition by DNA oligonucleotides after Exposure to Photoresists and Resist Removers

PubMed Central

Dean, Stacey L.; Morrow, Thomas J.; Patrick, Sue; Li, Mingwei; Clawson, Gary; Mayer, Theresa S.; Keating, Christine D.

2013-01-01

Combining biological molecules with integrated circuit technology is of considerable interest for next generation sensors and biomedical devices. Current lithographic microfabrication methods, however, were developed for compatibility with silicon technology rather than bioorganic molecules and consequently it cannot be assumed that biomolecules will remain attached and intact during on-chip processing. Here, we evaluate the effects of three common photoresists (Microposit S1800 series, PMGI SF6, and Megaposit SPR 3012) and two photoresist removers (acetone and 1165 remover) on the ability of surface-immobilized DNA oligonucleotides to selectively recognize their reverse-complementary sequence. Two common DNA immobilization methods were compared: adsorption of 5′-thiolated sequences directly to gold nanowires and covalent attachment of 5′-thiolated sequences to surface amines on silica coated nanowires. We found that acetone had deleterious effects on selective hybridization as compared to 1165 remover, presumably due to incomplete resist removal. Use of the PMGI photoresist, which involves a high temperature bake step, was detrimental to the later performance of nanowire-bound DNA in hybridization assays, especially for DNA attached via thiol adsorption. The other three photoresists did not substantially degrade DNA binding capacity or selectivity for complementary DNA sequences. To determine if the lithographic steps caused more subtle damage, we also tested oligonucleotides containing a single base mismatch. Finally, a two-step photolithographic process was developed and used in combination with dielectrophoretic nanowire assembly to produce an array of doubly-contacted, electrically isolated individual nanowire components on a chip. Post-fabrication fluorescence imaging indicated that nanowire-bound DNA was present and able to selectively bind complementary strands. PMID:23952639

On binding specificity of (6-4) photolyase to a T(6-4)T DNA photoproduct*

NASA Astrophysics Data System (ADS)

Jepsen, Katrine Aalbæk; Solov'yov, Ilia A.

2017-06-01

Different factors lead to DNA damage and if it is not repaired in due time, the damaged DNA could initiate mutagenesis and cancer. To avoid this deadly scenario, specific enzymes can scavenge and repair the DNA, but the enzymes have to bind first to the damaged sites. We have investigated this binding for a specific enzyme called (6-4) photolyase, which is capable of repairing certain UV-induced damage in DNA. Through molecular dynamics simulations we describe the binding between photolyase and the DNA and reveal that several charged amino acid residues in the enzyme, such as arginines and lysines turn out to be important. Especially R421 is crucial, as it keeps the DNA strands at the damaged site inside the repair pocket of the enzyme separated. DNA photolyase is structurally highly homologous to a protein called cryptochrome. Both proteins are biologically activated similarly, namely through flavin co-factor photoexcitation. It is, however, striking that cryptochrome cannot repair UV-damaged DNA. The present investigation allowed us to conclude on the small but, apparently, critical differences between photolyase and cryptochrome. The performed analysis gives insight into important factors that govern the binding of UV-damaged DNA and reveal why cryptochrome cannot have this functionality.

Fragmentation of DNA components by hyperthermal heavy ion (Ar+ and Xe+) impact in the condensed phase

NASA Astrophysics Data System (ADS)

Sarabipour, Sarvenaz; Sarvenaz Sarabipour, Ms; Michaud, Marc; Deng, Zongwu; Huels, Michael A.

The overriding environmental factor that presently limits human endeavors in space is exposure to heavy ion radiation. While knowledge of its damage to living tissue is essential for radiation protection and risk estimates for astronauts, very little data exists at the molecular level regarding the nascent DNA damage by the primary particle track, or by secondary species during subsequent reaction cascades. This persistent lack of a basic understanding of nascent damage induced by such low dose, high LET radiation, introduces unacceptable errors in radiation risk estimates (based mainly on extrapolation from high dose, low LET radiation), particularly for long term exposure. Mutagenic effects induced by heavy ion radiation to cells are largely due to DNA damage by secondary transient species, i.e. secondary ballistic ions, electrons and radicals generated along the ion tracks; the secondary ions have hyperthermal energies up to several 100 eV, which they will deposit within a few nm in the surrounding medium; thus their LET is very high, and yields lethal clustered DNA lesions. We present measurements of molecular damage induced in films of DNA components by ions with precisely such low energies (1-100 eV) and compare results to conventional electron impact measurements. Experiments are conducted in UHV using a mass selected low energy ion source, and a high-resolution quadrupole MS to monitor ion yields desorbing from molecular films. Among the major fragments, NH4 + is identified in the desorption mass spectra of irradiated films of Adenine, Guanine, Cytosine, indicating efficient deamination; in cells this results in pre-mutagenic lesions. Experiments with 5-amino-Uracil, and comparison to previous results on uracil and thymine show that deamination is a key step in the NH4 + fragment formation. For Adenine, we also observe formation of amine aducts in the films, viz. amination of Adenine, and global fragmentation in all ion impact mass spectra, attributed mainly to kinetic & potential ion scattering.[Funded by NSERC and the Canadian Space Agency].

Detection of ozone-induced DNA single strand breaks in murine bronchoalveolar lavage cells acutely exposed in vivo.

PubMed

Haney, J T; Connor, T H; Li, L

1999-04-01

Single-strand breaks (SSBs) in DNA have been used a biomarker of oxidative damage. The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of ozone (O(3)) to induce DNA SSBs in murine bronchoalveolar lavage (BAL) cells. The comet assay is more sensitive than other techniques currently utilized for detecting SSBs and requires fewer cells. In the present study, 3 mice were exposed for 3 h to 0.25 ppm of O(3), and 3 to 0.5 ppm of O(3) for 3 h. Two air-exposed mice served as negative controls. All mice were euthanized 3 h after exposure, at which time BAL cells were recovered from the lungs and stained with ethidium bromide. BAL cells recovered from an air-exposed mouse were exposed to various concentrations of H(2)O(2) in vitro for 1 h at 4 degrees C. Excluding cells from the H(2)O(2) group (n = 25), 50 randomly selected BAL cells were graded by comet tail length into 1 of 4 categories: no damage (0 mm), low damage (1-10 mm), medium damage (11-30 mm), and high damage (31 + mm). The nonparametric Wilcoxon rank-sum test was used for statistical analysis, and p values lower than .05 were considered significant. The H(2)O(2) and the 0.25 and 0.5 ppm O3 groups showed statistically significant increases in DNA SSBs as compared to air-exposed controls. The results of this study indicate that (1) O(3) induces DNA strand breaks in murine BAL cells at 0.25 and 0.5 ppm, as evidenced by statistically significant increases in the length of comet tails for O(3)-exposed groups, and (2) the comet assay can be used to assess O(3)-induced SSBs for in vivo exposures. Therefore, it has the potential as a biomarker for in vivo oxidant exposures.

Calculation on spectrum of direct DNA damage induced by low-energy electrons including dissociative electron attachment.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2017-03-01

In this work, direct DNA damage induced by low-energy electrons (sub-keV) is simulated using a Monte Carlo method. The characteristics of the present simulation are to consider the new mechanism of DNA damage due to dissociative electron attachment (DEA) and to allow determining damage to specific bases (i.e., adenine, thymine, guanine, or cytosine). The electron track structure in liquid water is generated, based on the dielectric response model for describing electron inelastic scattering and on a free-parameter theoretical model and the NIST database for calculating electron elastic scattering. Ionization cross sections of DNA bases are used to generate base radicals, and available DEA cross sections of DNA components are applied for determining DNA-strand breaks and base damage induced by sub-ionization electrons. The electron elastic scattering from DNA components is simulated using cross sections from different theoretical calculations. The resulting yields of various strand breaks and base damage in cellular environment are given. Especially, the contributions of sub-ionization electrons to various strand breaks and base damage are quantitatively presented, and the correlation between complex clustered DNA damage and the corresponding damaged bases is explored. This work shows that the contribution of sub-ionization electrons to strand breaks is substantial, up to about 40-70%, and this contribution is mainly focused on single-strand break. In addition, the base damage induced by sub-ionization electrons contributes to about 20-40% of the total base damage, and there is an evident correlation between single-strand break and damaged base pair A-T.

Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay

PubMed Central

2012-01-01

Background To investigate whether different conditions of DNA structure and radiation treatment could modify heterogeneity of response. Additionally to study variance as a potential parameter of heterogeneity for radiosensitivity testing. Methods Two-hundred leukocytes per sample of healthy donors were split into four groups. I: Intact chromatin structure; II: Nucleoids of histone-depleted DNA; III: Nucleoids of histone-depleted DNA with 90 mM DMSO as antioxidant. Response to single (I-III) and twice (IV) irradiation with 4 Gy and repair kinetics were evaluated using %Tail-DNA. Heterogeneity of DNA damage was determined by calculation of variance of DNA-damage (V) and mean variance (Mvar), mutual comparisons were done by one-way analysis of variance (ANOVA). Results Heterogeneity of initial DNA-damage (I, 0 min repair) increased without histones (II). Absence of histones was balanced by addition of antioxidants (III). Repair reduced heterogeneity of all samples (with and without irradiation). However double irradiation plus repair led to a higher level of heterogeneity distinguishable from single irradiation and repair in intact cells. Increase of mean DNA damage was associated with a similarly elevated variance of DNA damage (r = +0.88). Conclusions Heterogeneity of DNA-damage can be modified by histone level, antioxidant concentration, repair and radiation dose and was positively correlated with DNA damage. Experimental conditions might be optimized by reducing scatter of comet assay data by repair and antioxidants, potentially allowing better discrimination of small differences. Amount of heterogeneity measured by variance might be an additional useful parameter to characterize radiosensitivity. PMID:22520045

Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

PubMed Central

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

2013-01-01

Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. PMID:23523584

Individual variations in the correlation between erythemal threshold, UV-induced DNA damage and sun-burn cell formation.

PubMed

Heenen, M; Giacomoni, P U; Golstein, P

2001-10-01

A linear correlation between erythema intensity and DNA damage upon exposure to UV has not been firmly established. Many of the deleterious effects of UV exposure do occur after exposure to suberythemal doses. After DNA damage, cells undergo DNA repair. It is commonly accepted that when the burden of damage is beyond the repair capacities, the cell undergoes programmed cell death or apoptosis. The aim of this study is to quantify the amount of UV-induced DNA damage (estimated via the measurement of DNA repair or unscheduled DNA synthesis or UDS) and cellular damage (estimated via the determination of the density of sunburn cells or SBC). If DNA damage and erythema are correlated, similar intensity of UDS and similar density of SBC should be found in volunteers irradiated with a UV dose equal to two minimal erythema doses (MED). Our results show that in 15 different individuals the same relative dose (2 MEDs) provokes UDS values, which vary within a factor of 4. An even larger variability affects SBC counts after the same relative dose. When DNA damage or SBC are plotted versus the absolute dose (i.e. the dose expressed in J/m(2)), there is a rough correlation (with several exceptions) between dose and extent of UDS and SBC counts. It seems possible to divide the volunteers into two subpopulations with different susceptibilities to UV damage. It is well known that UDS and SBC measurements are often affected by large experimental indeterminacy, yet, the analysis of our results makes it plausible to suggest that for the triggering of erythema, a common threshold value for DNA damage or for SBC count are not to be found. In conclusion, the erythema response seems to be loosely correlated with DNA damage. This suggests that the protection offered by the sunscreens against DNA damage, the molecular basis of UV-induced mutagenesis, might not be related to the sun protection factor (SPF) indicated on the label of sunscreens, which is evaluated using the erythema as an endpoint.

A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

PubMed Central

Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

2015-01-01

The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

A systematic analysis of factors localized to damaged chromatin reveals PARP-dependent recruitment of transcription factors

PubMed Central

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C.; Westbrook, Thomas F.; Harper, J. Wade; Elledge, Stephen J.

2015-01-01

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. PMID:26004182

Evaluation of environmental genotoxicity by comet assay in Columba livia.

PubMed

González-Acevedo, Anahi; García-Salas, Juan A; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Méndez-López, Luis F; Cortés-Gutiérrez, Elva I

2016-01-01

The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as "sentinels," as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD-FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.

PPARGC1A variation associated with DNA damage, diabetes, and cardiovascular diseases: the Boston Puerto Rican Health Study.

PubMed

Lai, Chao-Qiang; Tucker, Katherine L; Parnell, Laurence D; Adiconis, Xian; García-Bailo, Bibiana; Griffith, John; Meydani, Mohsen; Ordovás, José M

2008-04-01

Individuals with type 2 diabetes exhibit higher DNA damage and increased risk of cardiovascular disease (CVD). However, mechanisms underlying the association between DNA damage and development of type 2 diabetes and CVD are not understood. We sought to link peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PPARGC1A), a master transcriptional regulator of mitochondrial oxidative phosphorylation and cellular energy metabolism, with DNA damage, type 2 diabetes, and CVD. We measured DNA damage as urinary 8-hydroxydeoxyguanosine (8-OHdG) concentration and examined the relationship between nine PPARGC1A genetic variants, DNA damage, type 2 diabetes, and self-reported CVD in 959 participants of the Boston Puerto Rican Health Study. With respect to urinary 8-OHdG, PPARGC1A variants showed significant association, and PPARGC1A haplotypes exhibited significant association after correction for multiple testing. Two independent PPARGC1A variants associated significantly with type 2 diabetes (odds ratios [ORs] 1.35 and 2.46; P = 0.045 and <0.001). Carriers of minor alleles of two other PPARGC1A variants, both in strong linkage disequilibrium and associated with lower DNA damage, showed lower prevalence of CVD (ORs 0.53 and 0.65; P = 0.030 and 0.175). Moreover, we found that physical activity correlated negatively with DNA damage. It is plausible that low physical activity combined with risk haplotyes contribute to the high prevalence of type 2 diabetes in this population. We propose that PPARGC1A influences development of type 2 diabetes and CVD via DNA damage. Increasing physical activity, which induces PPARGC1A expression, is a potential strategy to slow DNA damage, thereby decreasing the risk of CVD for individuals with type 2 diabetes.

Escin-induced DNA damage promotes escin-induced apoptosis in human colorectal cancer cells via p62 regulation of the ATM/γH2AX pathway.

PubMed

Wang, Zhong; Chen, Qiang; Li, Bin; Xie, Jia-Ming; Yang, Xiao-Dong; Zhao, Kui; Wu, Yong; Ye, Zhen-Yu; Chen, Zheng-Rong; Qin, Zheng-Hong; Xing, Chun-Gen

2018-05-31

Escin, a triterpene saponin isolated from horse chestnut seed, has been used to treat encephaledema, tissue swelling and chronic venous insufficiency. Recent studies show that escin induces cell cycle arrest, tumor proliferation inhibition and tumor cell apoptosis. But the relationship between escin-induced DNA damage and cell apoptosis in tumor cells remains unclear. In this study, we investigated whether and how escin-induced DNA damage contributed to escin-induced apoptosis in human colorectal cancer cells. Escin (5-80 μg/mL) dose-dependently inhibited the cell viability and colony formation in HCT116 and HCT8 cells. Escin treatment induced DNA damage, leading to p-ATM and γH2AX upregulation. Meanwhile, escin treatment increased the expression of p62, an adaptor protein, which played a crucial role in controlling cell survival and tumorigenesis, and had a protective effect against escin-induced DNA damage: knockdown of p62 apparently enhanced escin-induced DNA damage, whereas overexpression of p62 reduced escin-induced DNA damage. In addition, escin treatment induced concentration- and time-dependent apoptosis. Similarly, knockdown of p62 significantly increased escin-induced apoptosis in vitro and produced en escin-like antitumor effect in vivo. Overexpression of p62 decreased the rate of apoptosis. Further studies revealed that the functions of p62 in escin-induced DNA damage were associated with escin-induced apoptosis, and p62 knockdown combined with the ATM inhibitor KU55933 augmented escin-induced DNA damage and further increased escin-induced apoptosis. In conclusion, our results demonstrate that p62 regulates ATM/γH2AX pathway-mediated escin-induced DNA damage and apoptosis.

Markers of oxidative DNA damage in human interventions with fruit and berries.

PubMed

Freese, Riitta

2006-01-01

Diets rich in fruit and vegetables are associated with a decreased risk of several cancers via numerous possible mechanisms. For example, phytochemicals may decrease oxidative DNA damage and enhance DNA repair. Markers of oxidative DNA damage in human dietary intervention trials used most frequently include oxidized nucleosides such as 7-hydro-8-oxo-2'-deoxyguanosine, which can be analyzed from isolated DNA or urine. Single-cell gel electrophoresis has been widely used to measure baseline or H2O2-induced DNA strand breaks or sites of modified bases sensitive to repair enzymes recognizing oxidized purines or pyrimidines. Recently, markers of DNA repair also have been used. Few controlled human dietary interventions have investigated the specific effects of fruit or berries. There are indications that kiwifruit can decrease H2O2 sensitivity of lymphocyte DNA ex vivo and enhance DNA repair. Carefully controlled studies with flavonoid-rich fruit or berry juices found only few significant differences; less rigorously controlled studies gave more optimistic results. Data on the effects of fruit and berries on DNA damage in humans are scarce and inconclusive; adequately controlled studies with validated markers are needed. Because levels of DNA damage are usually low in young healthy volunteers, groups with an enhanced risk of DNA damage should be studied.

First cytotoxic, genotoxic, and antigenotoxic assessment of Euterpe oleracea fruit oil (açaí) in cultured human cells.

PubMed

Marques, E S; Tsuboy, M S F; Carvalho, J C T; Rosa, P C P; Perazzo, F F; Gaivão, I O M; Maistro, E L

2017-08-17

Euterpe oleracea Mart., popularly known as "açaí", is a tropical fruit from the Amazon region where it has considerable economic importance. Açaí has been used as food and for several medicinal purposes. Despite the widespread use of this fruit, there is a lack of data regarding the safety of using this fruit oil exclusively. Therefore, we evaluated the in vitro cytotoxic, genotoxic, and antigenotoxic effects of E. oleracea fruit oil (EOO) in cultured human lymphocytes (non-metabolizing cells) and HepG2 cell line (human hepatoma) (metabolizing cells) by using MTT, comet, and micronucleus assays. A wide range of EOO concentrations was tested with a preliminary MTT assay, which allowed selecting five concentrations for comet and micronucleus assays: 2.5, 10, 100, 500, and 1000 µg/mL. The results showed that none of the EOO tested concentrations presented cytotoxic effects. The genotoxic assessment revealed an absence of significant DNA and chromosome damage in human lymphocytes and HepG2 cells but did not show chemoprotection against the DNA damage induced by methyl methanesulfonate and benzo[a]pyrene, used as DNA-damaging agents.

Mechanisms of cell killing by the new anti-cancer drug SR 4233

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, J.

SR 4233 (3-amino-1,2,4-benzotriazine, 1,4-dioxide) is a new potential anti-cancer drug which has a highly selective toxicity to hypoxic cells. This study investigated the mechanism of cell killing by this drug. Enzymatic studies have shown that SR 4233 is reductively metabolized to SR 4317 by the tumor cell lines SCVII and HT 1080 under hypoxic conditions. Cytochrome P-450 may play a major role in the reduction in both cell lines. DT diaphorase is the second most important enzyme in reducing SR 4233. In characterizing the major cellular target for SR 4233, the author has shown that damage to cell mitochondria ismore » produced largely under aerobic conditions, whereas DNA is likely to be the major target for cell death under hypoxic conditions. Further experiments demonstrated that DNA damage was similar to that produced by ionizing radiation at equitoxic doses, and chromosome aberrations can entirely account for cell death by SR 4233 under hypoxic conditions in the low dose range. Nevertheless, chromosome breaks produced by SR 4233 are less repairable than those produced by ionizing radiation, suggesting highly localized damage in the DNA by discrete foci of SR 4233 radicals.« less

Destabilization of the MutSα's protein-protein interface due to binding to the DNA adduct induced by anticancer agent carboplatin via molecular dynamics simulations.

PubMed

Negureanu, Lacramioara; Salsbury, Freddie R

2013-11-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα's surveillance for DNA errors would possibly be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations.

Curcumin causes DNA damage and affects associated protein expression in HeLa human cervical cancer cells.

PubMed

Shang, Hung-Sheng; Chang, Chuan-Hsun; Chou, Yu-Ru; Yeh, Ming-Yang; Au, Man-Kuan; Lu, Hsu-Feng; Chu, Yung-Lin; Chou, Hsiao-Min; Chou, Hsiu-Chen; Shih, Yung-Luen; Chung, Jing-Gung

2016-10-01

Cervical cancer is one of the most common cancers in women worldwide and it is a prominent cause of cancer mortality. Curcumin is one of the major compounds from Turmeric and has been shown to induce cytotoxic cell death in human cervical cancer cells. However, there is no study to show curcumin induced DNA damage action via the effect on the DNA damage and repair protein in cervical cancer cells in detail. In this study, we investigated whether or not curcumin induced cell death via DNA damage, chromatin condensation in human cervical cancer HeLa cells by using comet assay and DAPI staining, respectively, we found that curcumin induced cell death through the induction of DNA damage, and chromatin condensation. Western blotting and confocal laser microscopy examination were used to examine the effects of curcumin on protein expression associated with DNA damage, repair and translocation of proteins. We found that curcumin at 13 µM increased the protein levels associated with DNA damage and repair, such as O6-methylguanine-DNA methyltransferase, early-onset breast cancer 1 (BRCA1), mediator of DNA damage checkpoint 1, p-p53 and p-H2A.XSer140 in HeLa cells. Results from confocal laser systems microscopy indicated that curcumin increased the translocation of p-p53 and p-H2A.XSer140 from cytosol to nuclei in HeLa cells. In conclusion, curcumin induced cell death in HeLa cells via induction of DNA damage, and chromatin condensation in vitro.

DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

PubMed Central

Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

2011-01-01

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

Mechanisms of free radical-induced damage to DNA.

PubMed

Dizdaroglu, Miral; Jaruga, Pawel

2012-04-01

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY TOXIC INDUSTRIAL CHEMICALS

EPA Science Inventory

One of the reported effects for exposure to many of the toxic industrial chemicals is DNA damage. The present study describes a simple, rapid and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, ch...

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY STYRENE OXIDE

EPA Science Inventory

A rapid and simple assay to detect DNA damage to calf thymus DNA caused by styrene oxide (SO) is reported. This assay is based on changes observed in the melting and annealing behavior of the damaged DNA. The melting annealing process was monitored using a fluorescence indicat...

Virtual Screening for the Development of Dual-Inhibitors Targeting Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1.

PubMed

Cardamone, Francesca; Pizzi, Simone; Iacovelli, Federico; Falconi, Mattia; Desideri, Alessandro

2017-01-01

Human topoisomerase IB is an important target in cancer therapy and drugs selectively stabilizing the topoisomerase IB-DNA covalent complex are in clinical use for several cancer types. Tyrosyl- DNA phosphodiesterase 1 is involved in the DNA repair resolving the topoisomerase IB-DNA covalent complex that is extremely dangerous for the survival of the cells since it produces an irreversible DNA damage. Given the close biological relationship between these two enzymes, the development of synergistic inhibitors, called dual-inhibitors, is an important challenge in cancer therapy and computer-aided drug design may help in the identification of the best compounds. In this review, an overview of the compounds inhibiting one of the two enzymes or acting as dual inhibitors is provided. Moreover, the general procedures of the virtual screening approach, providing a description of two widely used opensource programs, namely AutoDock4 and AutoDock Vina, are described. Finally, an application of the two programs on a selected number of dual inhibitors for tyrosyl-DNA phosphodiesterase 1 and topoisomerase IB and their performance is briefly discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Green's Function Approach to Simulate DNA Damage by the Indirect Effect

NASA Technical Reports Server (NTRS)

Plante, Ianik; Cicinotta, Francis A.

2013-01-01

The DNA damage is of fundamental importance in the understanding of the effects of ionizing radiation. DNA is damaged by the direct effect of radiation (e.g. direct ionization) and by indirect effect (e.g. damage by.OH radicals created by the radiolysis of water). Despite years of research, many questions on the DNA damage by ionizing radiation remains. In the recent years, the Green's functions of the diffusion equation (GFDE) have been used extensively in biochemistry [1], notably to simulate biochemical networks in time and space [2]. In our future work on DNA damage, we wish to use an approach based on the GFDE to refine existing models on the indirect effect of ionizing radiation on DNA. To do so, we will use the code RITRACKS [3] developed at the NASA Johnson Space Center to simulate the radiation track structure and calculate the position of radiolytic species after irradiation. We have also recently developed an efficient Monte-Carlo sampling algorithm for the GFDE of reversible reactions with an intermediate state [4], which can be modified and adapted to simulate DNA damage by free radicals. To do so, we will use the known reaction rate constants between radicals (OH, eaq, H,...) and the DNA bases, sugars and phosphates and use the sampling algorithms to simulate the diffusion of free radicals and chemical reactions with DNA. These techniques should help the understanding of the contribution of the indirect effect in the formation of DNA damage and double-strand breaks.

DNA Damage Response, Redox Status and Hematopoiesis

PubMed Central

Weiss, Cary N.; Ito, Keisuke

2013-01-01

The ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into progenitors is essential for homeostasis of the hematopoietic system. The longevity of HSCs makes them vulnerable to accumulating DNA damage, which may be leukemogenic or result in senescence and cell death. Additionally, the ability of HSCs to self-renew and differentiate allows DNA damage to spread throughout the hematologic system, leaving the organism vulnerable to disease. In this review we discuss cell fate decisions made in the face of DNA damage and other cellular stresses, and the role of reactive oxygen species in the long-term maintenance of HSCs and their DNA damage response. PMID:24041596

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Satyender; Kumar, Vivek; Vashisht, Kapil

2011-11-15

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activitymore » toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 {+-} 2.15 vs. 6.24 {+-} 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: Black-Right-Pointing-Pointer Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. Black-Right-Pointing-Pointer Workers exposed to some OPs demonstrated increased DNA damage. Black-Right-Pointing-Pointer CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. Black-Right-Pointing-Pointer Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.« less

Formation of Clustered DNA Damage after High-LET Irradiation: A Review

NASA Technical Reports Server (NTRS)

Hada, Megumi; Georgakilas, Alexandros G.

2008-01-01

Radiation can cause as well as cure cancer. The risk of developing radiation-induced cancer has traditionally been estimated from cancer incidence among survivors of the atomic bombs in Hiroshima and Nagasaki. These data provide the best estimate of human cancer risk over the dose range for low linear energy transfer (LET) radiations, such as X- or gamma-rays. The situation of estimating the real biological effects becomes even more difficult in the case of high LET particles encountered in space or as the result of domestic exposure to particles from radon gas emitters or other radioactive emitters like uranium-238. Complex DNA damage, i.e., the signature of high-LET radiations comprises by closely spaced DNA lesions forming a cluster of DNA damage. The two basic groups of complex DNA damage are double strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions (OCDL). Theoretical analysis and experimental evidence suggest there is increased complexity and severity of complex DNA damage with increasing LET (linear energy transfer) and a high mutagenic or carcinogenic potential. Data available on the formation of clustered DNA damage (DSBs and OCDL) by high-LET radiations are often controversial suggesting a variable response to dose and type of radiation. The chemical nature and cellular repair mechanisms of complex DNA damage have been much less characterized than those of isolated DNA lesions like an oxidized base or a single strand break especially in the case of high-LET radiation. This review will focus on the induction of clustered DNA damage by high-LET radiations presenting the earlier and recent relative data.

Responses of chub (Leuciscus cephalus) populations to chemical stress, assessed by genetic markers, DNA damage and cytochrome P4501A induction.

PubMed

Larno, V; Laroche, J; Launey, S; Flammarion, P; Devaux, A

2001-06-01

Indicators of effects at the population level (genetic variation using allozymes) and early indicators of pollution (EROD activity and DNA strand break formation) were analysed in chub (Leuciscus cephalus) living in weakly and heavily contaminated stations of the Rhône River watershed. The genetic erosion was mainly detected in a fish population living in a contaminated small river system, through modifications in allelic and genotypic frequencies for PGM-2 locus and could be linked to a genetic bottleneck and to the reduced gene flow from upstream unable to maintain or restore the genetic diversity. In a contaminated large river system, the genetic diversity for PGM-2 and other loci was maintained and was probably the consequence of a high gene flow from upstream, linked to a sustained drift of larvae and juveniles in the system. A convergent increase of the frequency of the 90 allele at PGM-2 was observed in two contaminated stations compared with the reference station, this trend being confirmed on a more extensive geographic scale over the Rhône River basin. A high level of EROD activity was detected in both contaminated sites but only the fish in the large river system showed a significant DNA damage level compared to the reference population. The low DNA damage level and high hepato-somatic ratio characterized the impacted population of the small river system and could be associated to a chronic high-level exposure of fish to pollutants which selected individuals exhibiting a high level of DNA damage repair. In the two contaminated systems, some genotypes at the PGM-2 and EST-2 loci showed a low level of DNA damage and/or a high EROD activity and may be considered as being tolerant to pollutants. A higher tolerance of the most heterozygous fish was also detected in the contaminated large system and confirmed that a high level of heterozygosity may be necessary for survival in such a system.

An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

PubMed

Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive

2015-06-01

Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Oxidation in the nucleotide pool, the DNA damage response and cellular senescence: Defective bricks build a defective house.

PubMed

Rai, Priyamvada

2010-11-28

Activation of persistent DNA damage response (DDR) signaling is associated with the induction of a permanent proliferative arrest known as cellular senescence, a phenomenon intrinsically linked to both tissue aging as well as tumor suppression. The DNA damage observed in senescent cells has been attributed to elevated levels of reactive oxygen species (ROS), failing DNA damage repair processes, and/or oncogenic activation. It is not clear how labile molecules such as ROS are able to damage chromatin-bound DNA to a sufficient extent to invoke persistent DNA damage and DDR signaling. Recent evidence suggests that the nucleotide pool is a significant target for oxidants and that oxidized nucleotides, once incorporated into genomic DNA, can lead to the induction of a DNA strand break-associated DDR that triggers senescence in normal cells and in cells sustaining oncogene activation. Evasion of this DDR and resulting senescence is a key step in tumor progression. This review will explore the role of oxidation in the nucleotide pool as a major effector of oxidative stress-induced genotoxic damage and DDR in the context of cellular senescence and tumorigenic transformation. 2010 Elsevier B.V. All rights reserved.

Vitamin E Modifies High-Fat Diet-Induced Increase of DNA Strand Breaks, and Changes in Expression and DNA Methylation of Dnmt1 and MLH1 in C57BL/6J Male Mice.

PubMed

Remely, Marlene; Ferk, Franziska; Sterneder, Sonja; Setayesh, Tahereh; Kepcija, Tatjana; Roth, Sylvia; Noorizadeh, Rahil; Greunz, Martina; Rebhan, Irene; Wagner, Karl-Heinz; Knasmüller, Siegfried; Haslberger, Alexander

2017-06-14

Obesity is associated with low-grade inflammation, increased ROS production and DNA damage. Supplementation with antioxidants might ameliorate DNA damage and support epigenetic regulation of DNA repair. C57BL/6J male mice were fed a high-fat (HFD) or a control diet (CD) with and without vitamin E supplementation (4.5 mg/kg body weight (b.w.)) for four months. DNA damage, DNA promoter methylation and gene expression of Dnmt1 and a DNA repair gene ( MLH1 ) were assayed in liver and colon. The HFD resulted in organ specific changes in DNA damage, the epigenetically important Dnmt1 gene, and the DNA repair gene MLH1 . Vitamin E reduced DNA damage and showed organ-specific effects on MLH1 and Dnmt1 gene expression and methylation. These results suggest that interventions with antioxidants and epigenetic active food ingredients should be developed as an effective prevention for obesity-and oxidative stress-induced health risks.

Hide and seek: How do DNA glycosylases locate oxidatively damaged DNA bases amidst a sea of undamaged bases?

PubMed

Lee, Andrea J; Wallace, Susan S

2017-06-01

The first step of the base excision repair (BER) pathway responsible for removing oxidative DNA damage utilizes DNA glycosylases to find and remove the damaged DNA base. How glycosylases find the damaged base amidst a sea of undamaged bases has long been a question in the BER field. Single molecule total internal reflection fluorescence microscopy (SM TIRFM) experiments have allowed for an exciting look into this search mechanism and have found that DNA glycosylases scan along the DNA backbone in a bidirectional and random fashion. By comparing the search behavior of bacterial glycosylases from different structural families and with varying substrate specificities, it was found that glycosylases search for damage by periodically inserting a wedge residue into the DNA stack as they redundantly search tracks of DNA that are 450-600bp in length. These studies open up a wealth of possibilities for further study in real time of the interactions of DNA glycosylases and other BER enzymes with various DNA substrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Assessment of Multiple Types of DNA Damage in Human Placentas from Smoking and Non-smoking Women in the Czech Republic

PubMed Central

Margaret Pratt, M.; King, Leon C.; Adams, Linda D.; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A.; Manchester, David K.; Sram, Radim J.; DeMarini, David M.; Poirier, Miriam C.

2010-01-01

Three classes of DNA damage were assessed in human placentas collected (in 2000-4) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by 32P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49–312 PAH-DNA adducts/108 nucleotides, were found by IHC/ACIS in 14 immediately-fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7 – 8.6 stable/bulky DNA adducts/108 nucleotides and 0.6 – 47.2 AB sites/105 nucleotides. For all methods there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and non-smokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. PMID:20839217

Assessment of multiple types of DNA damage in human placentas from smoking and nonsmoking women in the Czech Republic.

PubMed

Pratt, M Margaret; King, Leon C; Adams, Linda D; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A; Manchester, David K; Sram, Radim J; DeMarini, David M; Poirier, Miriam C

2011-01-01

Three classes of DNA damage were assessed in human placentas collected (2000-2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by (32)P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49-312 PAH-DNA adducts/10(8) nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7-8.6 stable/bulky DNA adducts/10(8) nucleotides and 0.6-47.2 AB sites/10(5) nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. Copyright © 2010 Wiley-Liss, Inc.

MGMT hypomethylation is associated with DNA damage in workers exposed to low-dose benzene.

PubMed

Li, Jie; Zhang, Xinjie; He, Zhini; Sun, Qing; Qin, Fei; Huang, Zhenlie; Zhang, Xiao; Sun, Xin; Liu, Linhua; Chen, Liping; Gao, Chen; Wang, Shan; Wang, Fangping; Li, Daochuan; Zeng, Xiaowen; Deng, Qifei; Wang, Qing; Zhang, Bo; Tang, Huanwen; Chen, Wen; Xiao, Yongmei

2017-07-01

This study aims to assess the effects of low-dose benzene on DNA damage and O 6 -methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers. We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay. The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p < 0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage. MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.

Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

PubMed

Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

2017-03-15

NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. © 2017. Published by The Company of Biologists Ltd.

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart.

PubMed

Gredilla, R; Barja, G; López-Torres, M

2001-10-01

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T4) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks. Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA. These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.

Fructose-Rich Diet Affects Mitochondrial DNA Damage and Repair in Rats.

PubMed

Cioffi, Federica; Senese, Rosalba; Lasala, Pasquale; Ziello, Angela; Mazzoli, Arianna; Crescenzo, Raffaella; Liverini, Giovanna; Lanni, Antonia; Goglia, Fernando; Iossa, Susanna

2017-03-24

Evidence indicates that many forms of fructose-induced metabolic disturbance are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage; however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are events involved in metabolic disease resulting from a fructose-rich diet. In the present study, we evaluated the degree of oxidative damage to liver mtDNA and its repair, in addition to the state of oxidative stress and antioxidant defense in the liver of rats fed a high-fructose diet. We used male rats feeding on a high-fructose or control diet for eight weeks. Our results showed an increase in mtDNA damage in the liver of rats fed a high-fructose diet and this damage, as evaluated by the expression of DNA polymerase γ, was not repaired; in addition, the mtDNA copy number was found to be significantly reduced. A reduction in the mtDNA copy number is indicative of impaired mitochondrial biogenesis, as is the finding of a reduction in the expression of genes involved in mitochondrial biogenesis. In conclusion, a fructose-rich diet leads to mitochondrial and mtDNA damage, which consequently may have a role in liver dysfunction and metabolic diseases.

Aging of hematopoietic stem cells: DNA damage and mutations?

PubMed

Moehrle, Bettina M; Geiger, Hartmut

2016-10-01

Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

PubMed

You, B-J; Wu, Y-C; Lee, C-L; Lee, H-Z

2014-03-01

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

Alterations of GSH and MDA levels and their association with bee venom-induced DNA damage in human peripheral blood leukocytes.

PubMed

Gajski, Goran; Domijan, Ana-Marija; Garaj-Vrhovac, Vera

2012-07-01

Bee venom (BV) has toxic effects in a variety of cell systems and oxidative stress has been proposed as a possible mechanism of its toxicity. This study investigated the in vitro effect of BV on glutathione (GSH) and malondialdehyde (MDA) levels, and their association with BV-induced DNA strand breaks and oxidative DNA damage in human peripheral blood leukocytes (HPBLs). Blood samples were treated with BV at concentrations ranging from 0.1 to 10 μg/ml over different lengths of time, and DNA damage in HPBLs was monitored with the alkaline and formamidopyrimidine glycoslyase (FPG)-modified comet assays, while GSH and MDA levels were determined in whole blood. Results showed a significant increase in overall DNA damage and FPG-sensitive sites in DNA of HPBLs exposed to BV compared with HPBLs from controls. An increase in DNA damage (assessed with both comet assays) was significantly associated with changes in MDA and GSH levels. When pretreated with N-acetyl-L-cysteine, a source of cysteine for the synthesis of the endogenous antioxidant GSH, a significant reduction of the DNA damaging effects of BV in HPBLs was noted. This suggests that oxidative stress is at least partly responsible for the DNA damaging effects of BV. Copyright © 2012 Wiley Periodicals, Inc.

Low-dose environmental radiation, DNA damage, and cancer: the possible contribution of psychological factors.

PubMed

Cwikel, Julie G; Gidron, Yori; Quastel, Michael

2010-01-01

Radiation causes DNA damage, increases risk of cancer, and is associated with psychological stress responses. This article proposes an evidence-based integrative model in which psychological factors could interact with radiation by either augmenting or moderating the adverse effects of radiation on DNA integrity and eventual tumorigenesis. Based on a review of the literature, we demonstrate the following: (1) the effects of low-dose radiation exposures on DNA integrity and on tumorigenesis; (2) the effects of low-dose radiation exposure on psychological distress; (3) the relationship between psychological factors and DNA damage; and (4) the possibility that psychological stress augments and that psychological resource variables moderate radiation-induced DNA damage and risk of cancer. The additional contribution of psychological processes to radiation-DNA damage-cancer relationships needs further study, and if verified, has clinical implications.

Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and beta-chlorolactic acid, using the single cell gel/comet assay.

PubMed

El Ramy, R; Ould Elhkim, M; Lezmi, S; Poul, J M

2007-01-01

3-monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is beta-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and beta-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. beta-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.

Skin photoprotection by natural polyphenols: anti-inflammatory, antioxidant and DNA repair mechanisms.

PubMed

Nichols, Joi A; Katiyar, Santosh K

2010-03-01

Epidemiological, clinical and laboratory studies have implicated solar ultraviolet (UV) radiation in various skin diseases including, premature aging of the skin and melanoma and non-melanoma skin cancers. Chronic UV radiation exposure-induced skin diseases or skin disorders are caused by the excessive induction of inflammation, oxidative stress and DNA damage, etc. The use of chemopreventive agents, such as plant polyphenols, to inhibit these events in UV-exposed skin is gaining attention. Chemoprevention refers to the use of agents that can inhibit, reverse or retard the process of these harmful events in the UV-exposed skin. A wide variety of polyphenols or phytochemicals, most of which are dietary supplements, have been reported to possess substantial skin photoprotective effects. This review article summarizes the photoprotective effects of some selected polyphenols, such as green tea polyphenols, grape seed proanthocyanidins, resveratrol, silymarin and genistein, on UV-induced skin inflammation, oxidative stress and DNA damage, etc., with a focus on mechanisms underlying the photoprotective effects of these polyphenols. The laboratory studies conducted in animal models suggest that these polyphenols have the ability to protect the skin from the adverse effects of UV radiation, including the risk of skin cancers. It is suggested that polyphenols may favorably supplement sunscreens protection, and may be useful for skin diseases associated with solar UV radiation-induced inflammation, oxidative stress and DNA damage.

ATM inhibition induces synthetic lethality and enhances sensitivity of PTEN-deficient breast cancer cells to cisplatin.

PubMed

Li, Ke; Yan, Huaying; Guo, Wenhao; Tang, Mei; Zhao, Xinyu; Tong, Aiping; Peng, Yong; Li, Qintong; Yuan, Zhu

2018-05-01

PTEN deficiency often causes defects in DNA damage repair. Currently, effective therapies for breast cancer are lacking. ATM is an attractive target for cancer treatment. Previous studies suggested a synthetic lethality between PTEN and PARP. However, the synthetically lethal interaction between PTEN and ATM in breast cancer has not been reported. Moreover, the mechanism remains elusive. Here, using KU-60019, an ATM kinase inhibitor, we investigated ATM inhibition as a synthetically lethal strategy to target breast cancer cells with PTEN defects. We found that KU-60019 preferentially sensitizes PTEN-deficient MDA-MB-468 breast cancer cells to cisplatin, though it also slightly enhances sensitivity of PTEN wild-type breast cancer cells. The increased cytotoxic sensitivity is associated with apoptosis, as evidenced by flow cytometry and PARP cleavage. Additionally, the increase of DNA damage accumulation due to the decreased capability of DNA repair, as indicated by γ-H2AX and Rad51 foci, also contributed to this selective cytotoxicity. Mechanistically, compared with PTEN wild-type MDA-MB-231 cells, PTEN-deficient MDA-MB-468 cells have lower level of Rad51, higher ATM kinase activity, and display the elevated level of DNA damage. Moreover, these differences could be further enlarged by cisplatin. Our findings suggest that ATM is a promising target for PTEN-defective breast cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Mus308 Processes Oxygen and Nitrogen Ethylation DNA Damage in Germ Cells of Drosophila

PubMed Central

Díaz-Valdés, Nancy; Comendador, Miguel A.; Sierra, L. María

2010-01-01

The D. melanogaster mus308 gene, highly conserved among higher eukaryotes, is implicated in the repair of cross-links and of O-ethylpyrimidine DNA damage, working in a DNA damage tolerance mechanism. However, despite its relevance, its possible role on the processing of different DNA ethylation damages is not clear. To obtain data on mutation frequency and on mutation spectra in mus308 deficient (mus308−) conditions, the ethylating agent diethyl sulfate (DES) was analysed in postmeiotic male germ cells. These data were compared with those corresponding to mus308 efficient conditions. Our results indicate that Mus308 is necessary for the processing of oxygen and N-ethylation damage, for the survival of fertilized eggs depending on the level of induced DNA damage, and for an influence of the DNA damage neighbouring sequence. These results support the role of mus308 in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end-joinig system. PMID:20936147

Chromosome territories reposition during DNA damage-repair response

PubMed Central

2013-01-01

Background Local higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored. Results Our report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells. Conclusions Thus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response. PMID:24330859

DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice.

PubMed

Wang, Degui; Yu, Tianyu; Liu, Yongqiang; Yan, Jun; Guo, Yingli; Jing, Yuhong; Yang, Xuguang; Song, Yanfeng; Tian, Yingxia

2016-12-02

Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. Copyright © 2016 Elsevier Inc. All rights reserved.

Genotoxic effect of ethacrynic acid and impact of antioxidants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, William M.; Hoffman, Jared D.; Loo, George, E-mail: g_loo@uncg.edu

It is known that ethacrynic acid (EA) decreases the intracellular levels of glutathione. Whether the anticipated oxidative stress affects the structural integrity of DNA is unknown. Therefore, DNA damage was assessed in EA-treated HCT116 cells, and the impact of several antioxidants was also determined. EA caused both concentration-dependent and time-dependent DNA damage that eventually resulted in cell death. Unexpectedly, the DNA damage caused by EA was intensified by either ascorbic acid or trolox. In contrast, EA-induced DNA damage was reduced by N-acetylcysteine and by the iron chelator, deferoxamine. In elucidating the DNA damage, it was determined that EA increased themore » production of reactive oxygen species, which was inhibited by N-acetylcysteine and deferoxamine but not by ascorbic acid and trolox. Also, EA decreased glutathione levels, which were inhibited by N-acetylcysteine. But, ascorbic acid, trolox, and deferoxamine neither inhibited nor enhanced the capacity of EA to decrease glutathione. Interestingly, the glutathione synthesis inhibitor, buthionine sulfoxime, lowered glutathione to a similar degree as EA, but no noticeable DNA damage was found. Nevertheless, buthionine sulfoxime potentiated the glutathione-lowering effect of EA and intensified the DNA damage caused by EA. Additionally, in examining redox-sensitive stress gene expression, it was found that EA increased HO-1, GADD153, and p21mRNA expression, in association with increased nuclear localization of Nrf-2 and p53 proteins. In contrast to ascorbic acid, trolox, and deferoxamine, N-acetylcysteine suppressed the EA-induced upregulation of GADD153, although not of HO-1. Overall, it is concluded that EA has genotoxic properties that can be amplified by certain antioxidants. - Highlights: • Ethacrynic acid (EA) caused cellular DNA damage. • EA-induced DNA damage was potentiated by ascorbic acid or trolox. • EA increased ROS production, not inhibited by ascorbic acid or trolox. • EA decreased glutathione levels, not prevented by ascorbic acid or trolox. • Buthionine sulfoxime intensified the DNA damage caused by EA.« less

Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors

PubMed Central

Sankar, Savita; Patterson, Ethan; Lewis, Emily M.; Waller, Laura E.; Tong, Caili; Dearborn, Joshua; Wozniak, David; Rubin, Joshua B.; Kroll, Kristen L.

2017-01-01

Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies. PMID:29234490

DNA Damage among Wood Workers Assessed with the Comet Assay

PubMed Central

Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

2016-01-01

Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

[Study on three kinds of gasoline oxygenates-induced DNA damage in mice fibroblasts].

PubMed

Song, Chonglin; Zhang, Zhifu; Chen, Xue; Zhang, Yanfeng; Wang, Chunhua; Liu, Keming

2002-10-01

To study DNA damage of three kinds of gasoline oxygenates. Single cell gel electrophoresis assay(Comet assay) was used to detect the damage effects of three gasoline oxygenates[methyl tertiary butyl ether(MTBE), ethanol anhydrous(EA) and dimethyl carbonate(DMC)] on DNA in L-929 mice fibroblasts. In certain concentation(37.500-150.000 mg/ml), MTBE could directly cause DNA damage of L-929 mice fibroblasts. There was obvious dose-effect relationship, i.e. when the concentration of MTBE was increased from 9.375 to 150.000 mg/ml, the comet rate also increased from 4% to 85%, and the length of comet tail changed correspondingly. The results of EA and DMC were negative. Under the condition of this experiment(150.000 mg/ml), MTBE could directly cause DNA damage while the effect of EA and DMC on DNA damage was not found.

DNA damage in blood cells exposed to low-level lasers.

PubMed

Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

2015-04-01

In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

ATM directs DNA damage responses and proteostasis via genetically separable pathways

PubMed Central

Lee, Ji-Hoon; Mand, Michael R.; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W.; Richards, Alicia L.; Coon, Joshua J.; Paull, Tanya T.

2018-01-01

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. Here, we genetically separated DNA damage activation of ATM from oxidative activation using separation-of-function mutations. We found that deficiency in ATM activation by Mre11-Rad50-Nbs1 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 ATM lacking oxidative activation generates 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 implicates ATM in the control of protein homeostasis. PMID:29317520

The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment.

PubMed

Lewis, Sheena E M; John Aitken, R; Conner, Sarah J; Iuliis, Geoffry De; Evenson, Donald P; Henkel, Ralph; Giwercman, Aleksander; Gharagozloo, Parviz

2013-10-01

Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. With all of these fertility check points, it shows more promise than conventional semen parameters from a diagnostic perspective. Despite this, few infertility clinics use it routinely. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths and weaknesses and clinical applicability of current sperm DNA fragmentation tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of increased oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. As those working in this field of clinical research, we conclude that DNA damage in human spermatozoa is an important attribute of semen quality which should be carefully assessed in the clinical work up of infertile couples and that properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6more » μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.« less

Destabilization of the MutSα’s protein-protein interface due to binding to the DNA adduct induced by anticancer agent Carboplatin via molecular dynamics simulations

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R

2013-01-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα’s surveillance for DNA errors would possible be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations. PMID:24061854

UV and ionizing radiations induced DNA damage, differences and similarities

NASA Astrophysics Data System (ADS)

Ravanat, Jean-Luc; Douki, Thierry

2016-11-01

Both UV and ionizing radiations damage DNA. Two main mechanisms, so-called direct and indirect pathways, are involved in the degradation of DNA induced by ionizing radiations. The direct effect of radiation corresponds to direct ionization of DNA (one electron ejection) whereas indirect effects are produced by reactive oxygen species generated through water radiolysis, including the highly reactive hydroxyl radicals, which damage DNA. UV (and visible) light damages DNA by again two distinct mechanisms. UVC and to a lesser extend UVB photons are directly absorbed by DNA bases, generating their excited states that are at the origin of the formation of pyrimidine dimers. UVA (and visible) light by interaction with endogenous or exogenous photosensitizers induce the formation of DNA damage through photosensitization reactions. The excited photosensitizer is able to induce either a one-electron oxidation of DNA (type I) or to produce singlet oxygen (type II) that reacts with DNA. In addition, through an energy transfer from the excited photosensitizer to DNA bases (sometime called type III mechanism) formation of pyrimidine dimers could be produced. Interestingly it has been shown recently that pyrimidine dimers are also produced by direct absorption of UVA light by DNA, even if absorption of DNA bases at these wavelengths is very low. It should be stressed that some excited photosensitizers (such as psoralens) could add directly to DNA bases to generate adducts. The review will described the differences and similarities in terms of damage formation (structure and mechanisms) between these two physical genotoxic agents.

Dose-rate effect of ultrashort electron beam radiation on DNA damage and repair in vitro.

PubMed

Babayan, Nelly; Hovhannisyan, Galina; Grigoryan, Bagrat; Grigoryan, Ruzanna; Sarkisyan, Natalia; Tsakanova, Gohar; Haroutiunian, Samvel; Aroutiounian, Rouben

2017-11-01

Laser-generated electron beams are distinguished from conventional accelerated particles by ultrashort beam pulses in the femtoseconds to picoseconds duration range, and their application may elucidate primary radiobiological effects. The aim of the present study was to determine the dose-rate effect of laser-generated ultrashort pulses of 4 MeV electron beam radiation on DNA damage and repair in human cells. The dose rate was increased via changing the pulse repetition frequency, without increasing the electron energy. The human chronic myeloid leukemia K-562 cell line was used to estimate the DNA damage and repair after irradiation, via the comet assay. A distribution analysis of the DNA damage was performed. The same mean level of initial DNA damages was observed at low (3.6 Gy/min) and high (36 Gy/min) dose-rate irradiation. In the case of low-dose-rate irradiation, the detected DNA damages were completely repairable, whereas the high-dose-rate irradiation demonstrated a lower level of reparability. The distribution analysis of initial DNA damages after high-dose-rate irradiation revealed a shift towards higher amounts of damage and a broadening in distribution. Thus, increasing the dose rate via changing the pulse frequency of ultrafast electrons leads to an increase in the complexity of DNA damages, with a consequent decrease in their reparability. Since the application of an ultrashort pulsed electron beam permits us to describe the primary radiobiological effects, it can be assumed that the observed dose-rate effect on DNA damage/repair is mainly caused by primary lesions appearing at the moment of irradiation. © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

PubMed

Bausinger, Julia; Speit, Günter

2014-11-01

The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells. Copyright © 2014 Elsevier B.V. All rights reserved.

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fishermore » 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96 h in medium containing DMSO ± 60 μM PM or KU 55933 (48 h; 10 nM). PM-induced activation of DNA damage repair genes was observed as early as 12 h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96 h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. - Highlights: • PM exposure induces DNA damage repair gene expression. • Inhibition of ATM prevented PM-induced follicle depletion. • PKCδ deficiency did not impact PM-induced ovotoxicity.« less

Chemical form of selenium differentially influences DNA repair pathways following exposure to lead nitrate.

PubMed

McKelvey, Shauna M; Horgan, Karina A; Murphy, Richard A

2015-01-01

Lead, an environmental toxin is known to induce a broad range of physiological and biochemical dysfunctions in humans through a number of mechanisms including the deactivation of antioxidants thus leading to generation of reactive oxygen species (ROS) and subsequent DNA damage. Selenium on the other hand has been proven to play an important role in the protection of cells from free radical damage and oxidative stress, though its effects are thought to be form and dose dependent. As the liver is the primary organ required for metabolite detoxification, HepG2 cells were chosen to assess the protective effects of various selenium compounds following exposure to the genotoxic agent lead nitrate. Initially DNA damage was quantified using a comet assay, gene expression patterns associated with DNA damage and signalling were also examined using PCR arrays and the biological pathways which were most significantly affected by selenium were identified. Interestingly, the organic type selenium compounds (selenium yeast and selenomethionine) conferred protection against lead induced DNA damage in HepG2 cells; this is evident by reduction in the quantity of DNA present in the comet tail of cells cultured in their presence with lead. This trend also followed through the gene expression changes noted in DNA damage pathways analysed. These results were in contrast with those of inorganic sodium selenite which promoted lead induced DNA damage evident in both the comet assay results and the gene expression analysis. Over all this study provided valuable insights into the effects which various selenium compounds had on the DNA damage and signalling pathway indicating the potential for using organic forms of selenium such as selenium enriched yeast to protect against DNA damaging agents. Copyright © 2014 Elsevier GmbH. All rights reserved.

Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways

PubMed Central

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-01-01

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms. PMID:16710445

Epigenetic telomere protection by Drosophila DNA damage response pathways.

PubMed

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-05-01

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

Ursolic acid exerts anti-cancer activity by suppressing vaccinia-related kinase 1-mediated damage repair in lung cancer cells.

PubMed

Kim, Seong-Hoon; Ryu, Hye Guk; Lee, Juhyun; Shin, Joon; Harikishore, Amaravadhi; Jung, Hoe-Yune; Jung, Hoe-Youn; Kim, Ye Seul; Lyu, Ha-Na; Oh, Eunji; Baek, Nam-In; Choi, Kwan-Yong; Yoon, Ho Sup; Kim, Kyong-Tai

2015-09-28

Many mitotic kinases have been targeted for the development of anti-cancer drugs, and inhibitors of these kinases have been expected to perform well for cancer therapy. Efforts focused on selecting good targets and finding specific drugs to target are especially needed, largely due to the increased frequency of anti-cancer drugs used in the treatment of lung cancer. Vaccinia-related kinase 1 (VRK1) is a master regulator in lung adenocarcinoma and is considered a key molecule in the adaptive pathway, which mainly controls cell survival. We found that ursolic acid (UA) inhibits the catalytic activity of VRK1 via direct binding to the catalytic domain of VRK1. UA weakens surveillance mechanisms by blocking 53BP1 foci formation induced by VRK1 in lung cancer cells, and possesses synergistic anti-cancer effects with DNA damaging drugs. Taken together, UA can be a good anti-cancer agent for targeted therapy or combination therapy with DNA damaging drugs for lung cancer patients.

ATM Deficiency Generating Genomic Instability Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Therapy-Induced DNA Damage.

PubMed

Perkhofer, Lukas; Schmitt, Anna; Romero Carrasco, Maria Carolina; Ihle, Michaela; Hampp, Stephanie; Ruess, Dietrich Alexander; Hessmann, Elisabeth; Russell, Ronan; Lechel, André; Azoitei, Ninel; Lin, Qiong; Liebau, Stefan; Hohwieler, Meike; Bohnenberger, Hanibal; Lesina, Marina; Algül, Hana; Gieldon, Laura; Schröck, Evelin; Gaedcke, Jochen; Wagner, Martin; Wiesmüller, Lisa; Sipos, Bence; Seufferlein, Thomas; Reinhardt, Hans Christian; Frappart, Pierre-Olivier; Kleger, Alexander

2017-10-15

Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial-mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576-90. ©2017 AACR . ©2017 American Association for Cancer Research.

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing.

PubMed

Hu, Jian-hong; Li, Qing-wang; Jiang, Zhong-liang; Li, Wen-ye

2008-12-01

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing-thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P<0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100mM trehalose, but cryopreservation could increase the degree of DNA damage (P<0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.

A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

PubMed Central

Moggs, Jonathan G.; Grandi, Paola; Quivy, Jean-Pierre; Jónsson, Zophonías O.; Hübscher, Ulrich; Becker, Peter B.; Almouzni, Geneviève

2000-01-01

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control. PMID:10648606

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

PubMed

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C; Westbrook, Thomas F; Harper, J Wade; Elledge, Stephen J

2015-06-09

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Mechanisms of DNA damage, repair and mutagenesis

PubMed Central

Chatterjee, Nimrat; Walker, Graham C.

2017-01-01

Living organisms are continuously exposed to a myriad of DNA damaging agents that can impact health and modulate disease-states. However, robust DNA repair and damage-bypass mechanisms faithfully protect the DNA by either removing or tolerating the damage to ensure an overall survival. Deviations in this fine-tuning are known to destabilize cellular metabolic homeostasis, as exemplified in diverse cancers where disruption or deregulation of DNA repair pathways results in genome instability. Because routinely used biological, physical and chemical agents impact human health, testing their genotoxicity and regulating their use have become important. In this introductory review, we will delineate mechanisms of DNA damage and the counteracting repair/tolerance pathways to provide insights into the molecular basis of genotoxicity in cells that lays the foundation for subsequent articles in this issue. PMID:28485537

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution.

PubMed

Gahlon, Hailey L; Romano, Louis J; Rueda, David

2017-11-20

Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.

A survey of the sequence-specific interaction of damaging agents with DNA: emphasis on antitumor agents.

PubMed

Murray, V

1999-01-01

This article reviews the literature concerning the sequence specificity of DNA-damaging agents. DNA-damaging agents are widely used in cancer chemotherapy. It is important to understand fully the determinants of DNA sequence specificity so that more effective DNA-damaging agents can be developed as antitumor drugs. There are five main methods of DNA sequence specificity analysis: cleavage of end-labeled fragments, linear amplification with Taq DNA polymerase, ligation-mediated polymerase chain reaction (PCR), single-strand ligation PCR, and footprinting. The DNA sequence specificity in purified DNA and in intact mammalian cells is reviewed for several classes of DNA-damaging agent. These include agents that form covalent adducts with DNA, free radical generators, topoisomerase inhibitors, intercalators and minor groove binders, enzymes, and electromagnetic radiation. The main sites of adduct formation are at the N-7 of guanine in the major groove of DNA and the N-3 of adenine in the minor groove, whereas free radical generators abstract hydrogen from the deoxyribose sugar and topoisomerase inhibitors cause enzyme-DNA cross-links to form. Several issues involved in the determination of the DNA sequence specificity are discussed. The future directions of the field, with respect to cancer chemotherapy, are also examined.

Increased oxidative phosphorylation in response to acute and chronic DNA damage

PubMed Central

Brace, Lear E; Vose, Sarah C; Stanya, Kristopher; Gathungu, Rose M; Marur, Vasant R; Longchamp, Alban; Treviño-Villarreal, Humberto; Mejia, Pedro; Vargas, Dorathy; Inouye, Karen; Bronson, Roderick T; Lee, Chih-Hao; Neilan, Edward; Kristal, Bruce S; Mitchell, James R

2016-01-01

Accumulation of DNA damage is intricately linked to aging, aging-related diseases and progeroid syndromes such as Cockayne syndrome (CS). Free radicals from endogenous oxidative energy metabolism can damage DNA, however the potential of acute or chronic DNA damage to modulate cellular and/or organismal energy metabolism remains largely unexplored. We modeled chronic endogenous genotoxic stress using a DNA repair-deficient Csa−/−|Xpa−/− mouse model of CS. Exogenous genotoxic stress was modeled in mice in vivo and primary cells in vitro treated with different genotoxins giving rise to diverse spectrums of lesions, including ultraviolet radiation, intrastrand crosslinking agents and ionizing radiation. Both chronic endogenous and acute exogenous genotoxic stress increased mitochondrial fatty acid oxidation (FAO) on the organismal level, manifested by increased oxygen consumption, reduced respiratory exchange ratio, progressive adipose loss and increased FAO in tissues ex vivo. In multiple primary cell types, the metabolic response to different genotoxins manifested as a cell-autonomous increase in oxidative phosphorylation (OXPHOS) subsequent to a transient decline in steady-state NAD+ and ATP levels, and required the DNA damage sensor PARP-1 and energy-sensing kinase AMPK. We conclude that increased FAO/OXPHOS is a general, beneficial, adaptive response to DNA damage on cellular and organismal levels, illustrating a fundamental link between genotoxic stress and energy metabolism driven by the energetic cost of DNA damage. Our study points to therapeutic opportunities to mitigate detrimental effects of DNA damage on primary cells in the context of radio/chemotherapy or progeroid syndromes. PMID:28721274

The comparison of DNA damage induced by micro DBD plasma and low energy electron for curing human diseases

NASA Astrophysics Data System (ADS)

Park, Yeunsoo

2015-09-01

It is well known that low energy electrons (LEE, especially below 10 eV) can generate DNA damage via indirect action named dissociative electron attachment (DEA). We can now explain some parts of the exact mechanism on DNA damage by LEE collision with direct ionization effect when cancer patients get the radiotherapy. It is kind of remarkable information in the field of radiation therapy. However, it is practically very difficult to directly apply this finding to human disease cure due to difficulty of LEE therapy actualization and request of further clinical studies. Recently, there is a novel challenge in plasma application, that is, how we can apply plasma technology to diagnosis and treatment of many serious diseases like cancer. Cold atmospheric pressure plasma (CAPP) is a very good source to apply to plasma medicine and bio-applications because of low temperature, low cost, and easy handling. Some scientists have already reported good results related to clinical plasma application. The purposes of this study are to further find out exact mechanisms of DNA damage by LEE at the molecular level, to verify new DNA damage like structural alteration on DNA subunits and to compare DNA damage by LEE and plasma source. We will keep expanding our study to DNA damage by plasma source to develop plasma-based new medical and biological applications. We will show some recent results, DNA damage by LEE and non-thermal plasma.

Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

PubMed

Morris, Lydia P; Conley, Andrew B; Degtyareva, Natalya; Jordan, I King; Doetsch, Paul W

2017-11-01

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

DNA Damage Signals and Space Radiation Risk

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.

2011-01-01

Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

In situ analysis of DNA damage response and repair using laser microirradiation.

PubMed

Kim, Jong-Soo; Heale, Jason T; Zeng, Weihua; Kong, Xiangduo; Krasieva, Tatiana B; Ball, Alexander R; Yokomori, Kyoko

2007-01-01

A proper response to DNA damage is critical for the maintenance of genome integrity. However, it is difficult to study the in vivo kinetics and factor requirements of the damage recognition process in mammalian cells. In order to address how the cell reacts to DNA damage, we utilized a second harmonic (532 nm) pulsed Nd:YAG laser to induce highly concentrated damage in a small area in interphase cell nuclei and cytologically analyzed both protein recruitment and modification. Our results revealed for the first time the sequential recruitment of factors involved in two major DNA double-strand break (DSB) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), and the cell cycle-specific recruitment of the sister chromatid cohesion complex cohesin to the damage site. In this chapter, the strategy developed to study the DNA damage response using the 532-nm Nd:YAG laser will be summarized.

Evaluating In Vitro DNA Damage Using Comet Assay.

PubMed

Lu, Yanxin; Liu, Yang; Yang, Chunzhang

2017-10-11

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

PubMed

Dutertre, Martin; Vagner, Stéphan

2017-10-27

Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intracellular iron overload leading to DNA damage of lymphocytes and immune dysfunction in thalassemia major patients.

PubMed

Shaw, Jyoti; Chakraborty, Ayan; Nag, Arijit; Chattopadyay, Arnab; Dasgupta, Anjan K; Bhattacharyya, Maitreyee

2017-11-01

To investigate the cause and effects of intracellular iron overload in lymphocytes of thalassemia major patients. Sixty-six thalassemia major patients having iron overload and 10 age-matched controls were chosen for the study. Blood sample was collected, and serum ferritin, oxidative stress; lymphocyte DNA damage were examined, and infective episodes were also counted. Case-control analysis revealed significant oxidative stress, iron overload, DNA damage, and rate of infections in thalassemia cases as compared to controls. For cases, oxidative stress (ROS) and iron overload (serum ferritin) showed good correlation with R 2  = 0.934 and correlation between DNA damage and ROS gave R 2  = 0.961. We also demonstrated that intracellular iron overload in thalassemia caused oxidative damage of lymphocyte DNA as exhibited by DNA damage assay. The inference is further confirmed by partial inhibition of such damage by chelation of iron and the concurrent lowering of the ROS level in the presence of chelator deferasirox. Therefore, intracellular iron overload caused DNA fragmentation, which may ultimately hamper lymphocyte function, and this may contribute to immune dysfunction and increased susceptibility to infections in thalassemia patients as indicated by the good correlation (R 2  = 0.91) between lymphocyte DNA damage and rate of infection found in this study. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Differences in DNA-damage in non-smoking men and women exposed to environmental tobacco smoke (ETS).

PubMed

Collier, Abby C; Dandge, Sachin D; Woodrow, James E; Pritsos, Chris A

2005-07-28

There is much data implicating environmental tobacco smoke (ETS) in the development and progression of disease, notably cancer, yet the mechanisms for this remain unclear. As ETS is both a pro-oxidant stressor and carcinogen, we investigated the relationship of ETS exposure to intracellular and serum levels of DNA-damage, both oxidative 8-hydroxy-2-deoxyguanosine (8OHdG) and general, in non-smokers from non-smoking households, occupationally exposed to ETS. General DNA-damage consisting of single and double strand breaks, alkali-labile sites and incomplete base-excision repair, increased significantly in a dose-dependent manner with ETS exposure in men (P=0.015, n=32, Pearson) but not women (P=0.736, n=17). Intracellular 8OHdG-DNA-damage and general DNA-damage were both greater in men than women (P=0.0005 and 0.016, respectively) but 8OHdG serum levels did not differ between the genders. Neither 8OHdG-DNA-damage nor serum levels correlated with increasing ETS exposure. This is the first study to demonstrate dose-dependent increases in DNA-damage from workplace ETS exposure. Perhaps most interesting was that despite equivalent ETS exposure, significantly greater DNA-damage occurred in men than women. These data may begin to provide a mechanistic rationale for the generally higher incidence of some diseases in males due to tobacco smoke and/or other genotoxic stressors.

Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

PubMed Central

Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

2015-01-01

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

Chk1 Promotes DNA Damage Response Bypass following Oxidative Stress in a Model of Hydrogen Peroxide-Associated Ulcerative Colitis through JNK Inactivation and Chromatin Binding.

PubMed

Reissig, Kathrin; Silver, Andrew; Hartig, Roland; Schinlauer, Antje; Walluscheck, Diana; Guenther, Thomas; Siedentopf, Sandra; Ross, Jochen; Vo, Diep-Khanh; Roessner, Albert; Poehlmann-Nitsche, Angela

2017-01-01

Dysregulation of c-Jun N -terminal kinase (JNK) activation promoted DNA damage response bypass and tumorigenesis in our model of hydrogen peroxide-associated ulcerative colitis (UC) and in patients with quiescent UC (QUC), UC-related dysplasia, and UC-related carcinoma (UC-CRC), thereby adapting to oxidative stress. In the UC model, we have observed features of oncogenic transformation: increased proliferation, undetected DNA damage, and apoptosis resistance. Here, we show that Chk1 was downregulated but activated in the acute and quiescent chronic phases. In both phases, Chk1 was linked to DNA damage response bypass by suppressing JNK activation following oxidative stress, promoting cell cycle progression despite DNA damage. Simultaneously, activated Chk1 was bound to chromatin. This triggered histone acetylation and the binding of histone acetyltransferases and transcription factors to chromatin. Thus, chromatin-immobilized activated Chk1 executed a dual function by suppressing DNA damage response and simultaneously inducing chromatin modulation. This caused undetected DNA damage and increased cellular proliferation through failure to transmit the appropriate DNA damage signal. Findings in vitro were corroborated by chromatin accumulation of activated Chk1, Ac-H3, Ac-H4, and c-Jun in active UC (AUC) in vivo. Targeting chromatin-bound Chk1, GCN5, PCAF, and p300/CBP could be a novel therapeutic strategy to prevent UC-related tumor progression.

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts.

PubMed

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2011-12-15

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities. Copyright © 2011 Elsevier Inc. All rights reserved.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

PubMed

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells

PubMed Central

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells. PMID:25892555

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

PubMed Central

Mirza, Sameer; Katafiasz, Bryan J.; Kumar, Rakesh; Wang, Jun; Mohibi, Shakur; Jain, Smrati; Gurumurthy, Channabasavaiah Basavaraju; Pandita, Tej K.; Dave, Bhavana J.; Band, Hamid; Band, Vimla

2012-01-01

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3−/− cells. Notably, Ada3−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3fl/fl and Ada3−/− cells confirmed higher residual DNA damage in Ada3−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability. PMID:23095635

High-Resolution Genome-Wide Analysis of Irradiated (UV and γ-Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

PubMed Central

St. Charles, Jordan; Hazkani-Covo, Einat; Yin, Yi; Andersen, Sabrina L.; Dietrich, Fred S.; Greenwell, Patricia W.; Malc, Ewa; Mieczkowski, Piotr; Petes, Thomas D.

2012-01-01

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote. PMID:22267500

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells.

PubMed

Liang, Shenxuan; Yin, Lei; Shengyang Yu, Kevin; Hofmann, Marie-Claude; Yu, Xiaozhong

2017-01-01

Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Tenuifolide B from Cinnamomum tenuifolium Stem Selectively Inhibits Proliferation of Oral Cancer Cells via Apoptosis, ROS Generation, Mitochondrial Depolarization, and DNA Damage.

PubMed

Chen, Chung-Yi; Yen, Ching-Yu; Wang, Hui-Ru; Yang, Hui-Ping; Tang, Jen-Yang; Huang, Hurng-Wern; Hsu, Shih-Hsien; Chang, Hsueh-Wei

2016-11-05

The development of drugs that selectively kill oral cancer cells but are less harmful to normal cells still provide several challenges. In this study, the antioral cancer effects of tenuifolide B (TFB), extracted from the stem of the plant Cinnamomum tenuifolium are evaluated in terms of their effects on cancer cell viability, cell cycle analysis, apoptosis, oxidative stress, and DNA damage. Cell viability of oral cancer cells (Ca9-22 and CAL 27) was found to be significantly inhibited by TFB in a dose-responsive manner in terms of ATP assay, yielding IC 50 = 4.67 and 7.05 μM (24 h), but are less lethal to normal oral cells (HGF-1). Dose-responsive increases in subG1 populations as well as the intensities of flow cytometry-based annexin V/propidium iodide (PI) analysis and pancaspase activity suggested that apoptosis was inducible by TFB in these two types of oral cancer cells. Pretreatment with the apoptosis inhibitor (Z-VAD-FMK) reduced the annexin V intensity of these two TFB-treated oral cancer cells, suggesting that TFB induced apoptosis-mediated cell death to oral cancer cells. Cleaved-poly (ADP-ribose) polymerase (PARP) and cleaved-caspases 3, 8, and 9 were upregulated in these two TFB-treated oral cancer cells over time but less harmful for normal oral HGF-1 cells. Dose-responsive and time-dependent increases in reactive oxygen species (ROS) and decreases in mitochondrial membrane potential (MitoMP) in these two TFB-treated oral cancer cells suggest that TFB may generate oxidative stress as measured by flow cytometry. N -acetylcysteine (NAC) pretreatment reduced the TFB-induced ROS generation and further validated that ROS was relevant to TFB-induced cell death. Both flow cytometry and Western blotting demonstrated that the DNA double strand marker γH2AX dose-responsively increased in TFB-treated Ca9-22 cells and time-dependently increased in two TFB-treated oral cancer cells. Taken together, we infer that TFB can selectively inhibit cell proliferation of oral cancer cells through apoptosis, ROS generation, mitochondrial membrane depolarization, and DNA damage.

Inhibiting Mitochondrial DNA Ligase IIIα Activates Caspase 1-Dependent Apoptosis in Cancer Cells.

PubMed

Sallmyr, Annahita; Matsumoto, Yoshihiro; Roginskaya, Vera; Van Houten, Bennett; Tomkinson, Alan E

2016-09-15

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1-dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431-41. ©2016 AACR. ©2016 American Association for Cancer Research.

DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

PubMed

Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

2017-01-25

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G 2 /M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in vitro cellular studies, here we demonstrate independent biological roles for the DDR kinases DNA-PKcs, ATM, and ATR during neurogenesis. We show that DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G 2 checkpoint control and apoptosis in proliferating cells. Conversely, ATM is critical for controlling apoptosis in immature noncycling neural cells after DNA damage. These data demonstrate functionally distinct, but cooperative, roles for each kinase in preserving genome stability in the nervous system. Copyright © 2017 the authors 0270-6474/17/370893-13$15.00/0.

Assessing the Fidelity of Ancient DNA Sequences Amplified From Nuclear Genes

PubMed Central

Binladen, Jonas; Wiuf, Carsten; Gilbert, M. Thomas P.; Bunce, Michael; Barnett, Ross; Larson, Greger; Greenwood, Alex D.; Haile, James; Ho, Simon Y. W.; Hansen, Anders J.; Willerslev, Eske

2006-01-01

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine → guanine and thymine → cytosine) and type 2 transitions (cytosine → thymine and guanine → adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences. PMID:16299392

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

PubMed Central

Mavragani, Ifigeneia V.; Nikitaki, Zacharenia; Souli, Maria P.; Aziz, Asef; Nowsheen, Somaira; Aziz, Khaled; Rogakou, Emmy

2017-01-01

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair. PMID:28718816

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis.

PubMed

Mavragani, Ifigeneia V; Nikitaki, Zacharenia; Souli, Maria P; Aziz, Asef; Nowsheen, Somaira; Aziz, Khaled; Rogakou, Emmy; Georgakilas, Alexandros G

2017-07-18

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15-20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent "danger" signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.

Hypothermia modulates the DNA damage response to ionizing radiation in human peripheral blood lymphocytes.

PubMed

Lisowska, Halina; Cheng, Lei; Sollazzo, Alice; Lundholm, Lovisa; Wegierek-Ciuk, Aneta; Sommer, Sylwester; Lankoff, Anna; Wojcik, Andrzej

2018-06-01

Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8 °C and 37 °C. To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A. A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8 °C as compared to 37 °C already after 30 minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes. Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents

PubMed Central

Proctor, Michael; Flaherty, Patrick; Jordan, Michael I; Arkin, Adam P; Davis, Ronald W; Nislow, Corey; Giaever, Guri

2005-01-01

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups. PMID:16121259

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

PubMed

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Extracellular self-DNA as a damage-associated molecular pattern (DAMP) that triggers self-specific immunity induction in plants.

PubMed

Duran-Flores, Dalia; Heil, Martin

2017-10-16

Mammals sense self or non-self extracellular or extranuclear DNA fragments (hereinafter collectively termed eDNA) as indicators of injury or infection and respond with immunity. We hypothesised that eDNA acts as a damage-associated molecular pattern (DAMP) also in plants and that it contributes to self versus non-self discrimination. Treating plants and suspension-cultured cells of common bean (Phaseolus vulgaris) with fragmented self eDNA (obtained from other plants of the same species) induced early, immunity-related signalling responses such as H 2 O 2 generation and MAPK activation, decreased the infection by a bacterial pathogen (Pseudomonas syringae) and increased an indirect defence to herbivores (extrafloral nectar secretion). By contrast, non-self DNA (obtained from lima bean, Phaseolus lunatus, and Acacia farnesiana) had significantly lower or no detectable effects. Only fragments below a size of 700 bp were active, and treating the eDNA preparation DNAse abolished its inducing effects, whereas treatment with RNAse or proteinase had no detectable effect. These findings indicate that DNA fragments, rather than small RNAs, single nucleotides or proteins, accounted for the observed effects. We suggest that eDNA functions a DAMP in plants and that plants discriminate self from non-self at a species-specific level. The immune systems of plants and mammals share multiple central elements, but further work will be required to understand the mechanisms and the selective benefits of an immunity response that is triggered by eDNA in a species-specific manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct observation of ultrafast-electron-transfer reactions unravels high effectiveness of reductive DNA damage

PubMed Central

Nguyen, Jenny; Ma, Yuhan; Luo, Ting; Bristow, Robert G.; Jaffray, David A.; Lu, Qing-Bin

2011-01-01

Both water and electron-transfer reactions play important roles in chemistry, physics, biology, and the environment. Oxidative DNA damage is a well-known mechanism, whereas the relative role of reductive DNA damage is unknown. The prehydrated electron (), a novel species of electrons in water, is a fascinating species due to its fundamental importance in chemistry, biology, and the environment. is an ideal agent to observe reductive DNA damage. Here, we report both the first in situ femtosecond time-resolved laser spectroscopy measurements of ultrafast-electron-transfer (UET) reactions of with various scavengers (KNO3, isopropanol, and dimethyl sulfoxide) and the first gel electrophoresis measurements of DNA strand breaks induced by and OH• radicals co-produced by two-UV-photon photolysis of water. We strikingly found that the yield of reductive DNA strand breaks induced by each is twice the yield of oxidative DNA strand breaks induced by each OH• radical. Our results not only unravel the long-standing mystery about the relative role of radicals in inducing DNA damage under ionizing radiation, but also challenge the conventional notion that oxidative damage is the main pathway for DNA damage. The results also show the potential of femtomedicine as a new transdisciplinary frontier and the broad significance of UET reactions of in many processes in chemistry, physics, biology, and the environment. PMID:21730183

Stress-induced DNA Damage biomarkers: Applications and limitations

NASA Astrophysics Data System (ADS)

Nikitaki, Zacharenia; Hellweg, Christine; Georgakilas, Alexandros; Ravanat, Jean-Luc

2015-06-01

A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes like replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damages play a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g. X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e. single, complex DNA lesions etc. that can be used as biomarkers. We critically compare DNA damage detection methods and their limitations. In addition to such DNA damage products, we suggest possible gene inductions that can be used to characterize responses to different types of stresses i.e. radiation, oxidative and replication stress, based on bioinformatic approaches and stringent meta-analysis of literature data.

Electrochemical detection of DNA damage induced by acrylamide and its metabolite at the graphene-ionic liquid-Nafion modified pyrolytic graphite electrode.

PubMed

Qiu, Yanyan; Qu, Xiangjin; Dong, Jing; Ai, Shiyun; Han, Ruixia

2011-06-15

A new electrochemical biosensor for directly detecting DNA damage induced by acrylamide (AA) and its metabolite was presented in this work. The graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was prepared, and then horseradish peroxidase (HRP) and natural double-stranded DNA were alternately assembled on the modified electrode by the layer-by-layer method. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)(n) film were characterized by electrochemical impedance spectroscopy. With the guanine signal in DNA as an indicator, the damage of DNA was detected by differential pulse voltammetry after PGE/graphene-ionic liquid-Nafion/(HRP/DNA)(n) was incubated in AA solution or AA+H(2)O(2) solution at 37°C. This method provides a new model to mimic and directly detect DNA damage induced by chemical pollutants and their metabolites in vitro. The results indicated that, in the presence of H(2)O(2), HRP was activated and catalyzed the transformation of AA to glycidamide, which could form DNA adducts and induce more serious damage of DNA than AA. In order to further verify these results, UV-vis spectrophotometry was also used to investigate DNA damage induced by AA and its metabolites in solution and the similar results were obtained. Copyright © 2011 Elsevier B.V. All rights reserved.

Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - Implication in modification of radiation damage

PubMed Central

Anuranjani; Bala, Madhu

2014-01-01

Whole body exposure to low linear energy transfer (LET) ionizing radiations (IRs) damages vital intracellular bio-molecules leading to multiple cellular and tissue injuries as well as pathophysiologies such as inflammation, immunosuppression etc. Nearly 70% of damage is caused indirectly by radiolysis of intracellular water leading to formation of reactive oxygen species (ROS) and free radicals and producing a state of oxidative stress. The damage is also caused by direct ionization of biomolecules. The type of radiation injuries is dependent on the absorbed radiation dose. Sub-lethal IR dose produces more of DNA base damages, whereas higher doses produce more DNA single strand break (SSBs), and double strand breaks (DSBs). The Nrf2-ARE pathway is an important oxidative stress regulating pathway. The DNA DSBs repair regulated by MRN complex, immunomodulation and inflammation regulated by HMGB1 and various types of cytokines are some of the key pathways which interact with each other in a complex manner and modify the radiation response. Because the majority of radiation damage is via oxidative stress, it is essential to gain in depth understanding of the mechanisms of Nrf2-ARE pathway and understand its interactions with MRN complex, HMGB1 and cytokines to increase our understanding on the radiation responses. Such information is of tremendous help in development of medical radiation countermeasures, radioprotective drugs and therapeutics. Till date no approved and safe countermeasure is available for human use. This study reviews the Nrf2-ARE pathway and its crosstalk with MRN-complex, HMGB1 and cytokines (TNF-a, IL-6, IFN-? etc.). An attempt is also made to review the modification of some of these pathways in presence of selected antioxidant radioprotective compounds or herbal extracts. PMID:25009785

Upregulated ATM gene expression and activated DNA crosslink-induced damage response checkpoint in Fanconi anemia: implications for carcinogenesis.

PubMed

Yamamoto, Kazuhiko; Nihrane, Abdallah; Aglipay, Jason; Sironi, Juan; Arkin, Steven; Lipton, Jeffrey M; Ouchi, Toru; Liu, Johnson M

2008-01-01

Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects, leukemia, and squamous cell carcinoma of the head and neck (HNSCC) and cervix. The FA/BRCA pathway includes 8 members of a core complex and 5 downstream gene products closely linked with BRCA1 or BRCA2. Precancerous lesions are believed to trigger the DNA damage response (DDR), and we focused on the DDR in FA and its putative role as a checkpoint barrier to cancer. In primary fibroblasts with mutations in the core complex FANCA protein, we discovered that basal expression and phosphorylation of ATM (ataxia telangiectasia mutated) and p53 induced by irradiation (IR) or mitomycin C (MMC) were upregulated. This heightened response appeared to be due to increased basal levels of ATM in cultured FANCA-mutant cells, highlighting the new observation that ATM can be regulated at the transcriptional level in addition to its well-established activation by autophosphorylation. Functional analysis of this response using gamma-H2AX foci as markers of DNA double-stranded breaks (DSBs) demonstrated abnormal persistence of only MMC- and not IR-induced foci. Thus, we describe a processing defect that leads to general DDR upregulation but specific persistence of DNA crosslinker-induced damage response foci. Underscoring the significance of these findings, we found resistance to DNA crosslinker-induced cell cycle arrest and apoptosis in a TP53-mutant, patient-derived HNSCC cell line, whereas a lymphoblastoid cell line derived from this same individual was not mutated at TP53 and retained DNA crosslinker sensitivity. Our results suggest that cancer in FA may arise from selection for cells that escape from a chronically activated DDR checkpoint.

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site.

PubMed

Abe, Yukiko; Nakagawa, Osamu; Yamaguchi, Rie; Sasaki, Shigeki

2012-06-01

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings.

PubMed

Di Pietro, Angela; Visalli, Giuseppa; La Maestra, Sebastiano; Micale, Rosanna; Baluce, Barbara; Matarese, Giovanni; Cingano, Luciano; Scoglio, Maria Elena

2008-02-29

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.

New insights in the bacterial spore resistance to extreme terrestrial and extraterrestrial factors

NASA Astrophysics Data System (ADS)

Moeller, Ralf; Horneck, Gerda; Reitz, Guenther

Based on their unique resistance to various space parameters, Bacillus endospores are one of the model systems used for astrobiological studies. The extremely high resistance of bacterial endospores to environmental stress factors has intrigued researchers since long time and many characteristic spore features, especially those involved in the protection of spore DNA, have already been uncovered. The disclosure of the complete genomic sequence of Bacillus subtilis 168, one of the often used astrobiological model system, and the rapid development of tran-scriptional microarray techniques have opened new opportunities of gaining further insights in the enigma of spore resistance. Spores of B. subtilis were exposed to various extreme ter-restrial and extraterrestrial stressors to reach a better understanding of the DNA protection and repair strategies, which them to cope with the induced DNA damage. Following physical stress factors of environmental importance -either on Earth or in space -were selected for this thesis: (i) mono-and polychromatic UV radiation, (ii) ionizing radiation, (iii) exposure to ultrahigh vacuum; and (iv) high shock pressures simulating meteorite impacts. To reach a most comprehensive understanding of spore resistance to those harsh terrestrial or simulated extraterrestrial conditions, a standardized experimental protocol of the preparation and ana-lyzing methods was established including the determination of the following spore responses: (i) survival, (ii) induced mutations, (iii) DNA damage, (iv) role of different repair pathways by use of a set of repair deficient mutants, and (v) transcriptional responses during spore germi-nation by use of genome-wide transcriptome analyses and confirmation by RT-PCR. From this comprehensive set of data on spore resistance to a variety of environmental stress parameters a model of a "built-in" transcriptional program of bacterial spores in response to DNA damaging treatments to ensure DNA restoration during germination has been developed.

The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

PubMed

Agarwal, Poonam; Miller, Kyle M

2016-10-01

DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

PubMed

Carreras Puigvert, Jordi; von Stechow, Louise; Siddappa, Ramakrishnaiah; Pines, Alex; Bahjat, Mahnoush; Haazen, Lizette C J M; Olsen, Jesper V; Vrieling, Harry; Meerman, John H N; Mullenders, Leon H F; van de Water, Bob; Danen, Erik H J

2013-01-22

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

Imaging and radiation effects of gold nanoparticles in tumour cells

PubMed Central

McQuaid, Harold N.; Muir, Mark F.; Taggart, Laura E.; McMahon, Stephen J.; Coulter, Jonathan A.; Hyland, Wendy B.; Jain, Suneil; Butterworth, Karl T.; Schettino, Giuseppe; Prise, Kevin M.; Hirst, David G.; Botchway, Stanley W.; Currell, Fred J.

2016-01-01

Gold nanoparticle radiosensitization represents a novel technique in enhancement of ionising radiation dose and its effect on biological systems. Variation between theoretical predictions and experimental measurement is significant enough that the mechanism leading to an increase in cell killing and DNA damage is still not clear. We present the first experimental results that take into account both the measured biodistribution of gold nanoparticles at the cellular level and the range of the product electrons responsible for energy deposition. Combining synchrotron-generated monoenergetic X-rays, intracellular gold particle imaging and DNA damage assays, has enabled a DNA damage model to be generated that includes the production of intermediate electrons. We can therefore show for the first time good agreement between the prediction of biological outcomes from both the Local Effect Model and a DNA damage model with experimentally observed cell killing and DNA damage induction via the combination of X-rays and GNPs. However, the requirement of two distinct models as indicated by this mechanistic study, one for short-term DNA damage and another for cell survival, indicates that, at least for nanoparticle enhancement, it is not safe to equate the lethal lesions invoked in the local effect model with DNA damage events. PMID:26787230

Protective effect of KI in mtDNA in porcine thyroid: comparison with KIO₃ and nDNA.

PubMed

Karbownik-Lewinska, Malgorzata; Stepniak, Jan; Milczarek, Magdalena; Lewinski, Andrzej

2015-03-01

Iodine, bivalent iron (Fe²⁺), and hydrogen peroxide (H₂O₂), all significantly affecting the red-ox balance, are required for thyroid hormone synthesis. Intracellular iodine excess (≥10⁻³ M) transiently blocks thyroid hormonogenesis (an adaptive mechanism called Wolff-Chaikoff effect). The aim of the study was to evaluate the effects of iodine, used as potassium iodide (KI) or potassium iodate (KIO₃), in concentrations corresponding to those typical for Wolff-Chaikoff effect, on the level of oxidative damage to nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) isolated from porcine thyroid under basal conditions and in the presence of Fenton reaction (Fe²⁺+H₂O₂ → Fe³⁺+(·)OH + OH⁻) substrates. Thyroid nDNA and mtDNA were incubated in the presence of either KI or KIO₃ (2.5-50 mM), without/with FeSO₄ (30 µM) + H₂O₂ (0.5 mM). Index of DNA damage, i.e., 8-oxo-7,8-dihydro-2'-deoxyguanosine, was measured by HPLC. Neither KI nor KIO₃ increased the basal level of 8-oxodG in both nDNA and mtDNA. KI-in all used concentrations-completely prevented the damaging effect of Fenton reaction substrates in mtDNA, and it partially prevented this damage in nDNA. KIO₃ partially prevented Fe²⁺+H₂O₂-induced oxidative damage in both DNA only in its highest used concentrations (≥25 mM). Without additional prooxidative abuse, both iodine compounds, i.e., KI and KIO₃, seem to be safe in terms of their potential oxidative damage to DNA in the thyroid. The superiority of KI over KIO₃ relies on its stronger protective effects against oxidative damage to mtDNA, which constitutes an argument for its preferential utility in iodine prophylaxis.

MDC1: The art of keeping things in focus.

PubMed

Jungmichel, Stephanie; Stucki, Manuel

2010-08-01

The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures-usually termed DNA damage foci-is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structures.

DNA damage and repair after high LET radiation

NASA Astrophysics Data System (ADS)

O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

Cryopreservation of human blood for alkaline and Fpg-modified comet assay.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2016-01-01

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at -20 °C and -80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at -20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at -20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at -80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells†

PubMed Central

Ernst, Russell J.; Komor, Alexis C.; Barton, Jacqueline K.

2011-01-01

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents. PMID:22103240

The SOS Response is Permitted in Escherichia coli Strains Deficient in the Expression of the mazEF Pathway

DTIC Science & Technology

2014-12-03

DNA damage . It is controlled by a complex network involving the RecA and LexA proteins. We have previously shown that the SOS response to DNA damage ...Research Triangle Park, NC 27709-2211 enteric bacterium E. coli, SOS Response, DNA damage REPORT DOCUMENTATION PAGE 11. SPONSOR/MONITOR’S REPORT...Report Title The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized bacterial network induced by DNA damage

Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

PubMed

Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

2015-05-12

VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1

PubMed Central

Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

2017-01-01

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites. PMID:28325872

Characterization of a DNA damage-recognition protein mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Donahue, B.A.; Augot, M.; Bellon, S.F.

1990-06-19

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, K{sub d}, of the protein from cisplatin-modified DNA was estimated to be (1-20) {times} 10{sup {minus}10} M. Protein binding is selective for DNAmore » modified with cisplatin, (Pt(en)Cl{sub 2}) (en, ethylenediamine), and (Pt(dach)Cl{sub 2}) (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating (Pt(dien)Cl)Cl (dien, diethylenetriamine) complexes. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl{sub 2}, CdCl{sub 2}, CoCl{sub 2}, or ZnCl{sub 2} and with 1 mM HgCl{sub 2}. This protein, alone or in conjunction with other cellular constituents, could be of general importance in the initial stages of processing of mammalian DNA damaged by cisplatin or other genotoxic agents and may belong to a wider class of such cellular damage-recognition proteins (DRPs).« less

Inhibition of DNA-dependent protein kinase catalytic subunit by small molecule inhibitor NU7026 sensitizes human leukemic K562 cells to benzene metabolite-induced apoptosis.

PubMed

You, Hao; Kong, Meng-meng; Wang, Li-ping; Xiao, Xiao; Liao, Han-lin; Bi, Zhuo-yue; Yan, Hong; Wang, Hong; Wang, Chun-hong; Ma, Qiang; Liu, Yan-qun; Bi, Yong-yi

2013-02-01

Benzene is an established leukotoxin and leukemogen in humans. We have previously reported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate the cellular response to DNA double strand break (DSB) caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

PubMed Central

Park, Sojin; Choi, Seoyun; Ahn, Byungchan

2016-01-01

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

PubMed Central

Hawkins, Clare L; Pattison, David I; Davies, Michael J

2002-01-01

Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines. These results are consistent with the predictions of the kinetic model and suggest that histones are major targets for HOCl in the nucleosome. Furthermore, the resulting protein chloramines and the radicals derived from them may act as contributing agents in HOCl-mediated DNA oxidation. PMID:12010123

Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

PubMed

Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu

2014-07-15

The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.

PubMed

Takada, Saeko; Collins, Eric R; Kurahashi, Kayo

2015-05-15

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage. © 2015 Takada et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

LRRK2 interacts with ATM and regulates Mdm2-p53 cell proliferation axis in response to genotoxic stress.

PubMed

Chen, Zhongcan; Cao, Zhen; Zhang, Wei; Gu, Minxia; Zhou, Zhi Dong; Li, Baojie; Li, Jing; Tan, Eng King; Zeng, Li

2017-11-15

Pathogenic leucine-rich repeat kinase 2 (LRRK2) mutations are recognized as the most common cause of familial Parkinson's disease in certain populations. Recently, LRRK2 mutations were shown to be associated with a higher risk of hormone-related cancers. However, how LRRK2 itself contributes to cancer risk remains unknown. DNA damage causes cancer, and DNA damage responses are among the most important pathways in cancer biology. To understand the role of LRRK2 in DNA damage response pathway, we induced DNA damage by applying genotoxic stress to the cells with Adriamycin. We found that DNA damage enhances LRRK2 phosphorylation at Serine 910, Serine 935 and Serine 1292. We further showed that LRRK2 phosphorylation is abolished in the absence of ATM, suggesting that LRRK2 phosphorylation requires ATM. It should also be noted that LRRK2 interacts with ATM. In contrast, overexpression or knockdown of LRRK2 does not affect ATM phosphorylation, indicating that LRRK2 is the downstream target of ATM in response to DNA damage. Moreover, we demonstrated that LRRK2 increases the expression of p53 and p21 by increasing the Mdm2 phosphorylation in response to DNA damage. Loss-of-function in LRRK2 has the opposite effect to that of LRRK2. In addition, FACS analysis revealed that LRRK2 enhances cell cycle progression into S phase in response to DNA damage, a finding that was confirmed by 5-bromo-2'-deoxyuridine immunostaining. Taken together, our findings demonstrate that LRRK2 plays an important role in the ATM-Mdm2-p53 pathway that regulates cell proliferation in response to DNA damage. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mitochondrial DNA damage and vascular function in patients with diabetes mellitus and atherosclerotic cardiovascular disease.

PubMed

Fetterman, Jessica L; Holbrook, Monica; Westbrook, David G; Brown, Jamelle A; Feeley, Kyle P; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Weisbrod, Robert M; Widlansky, Michael E; Gokce, Noyan; Ballinger, Scott W; Hamburg, Naomi M

2016-03-31

Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (β = 0.14 ± 0.13, P = 0.04 and β = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.

Alkaline ceramidase 2 and its bioactive product sphingosine are novel regulators of the DNA damage response

PubMed Central

Xu, Ruijuan; Wang, Kai; Mileva, Izolda; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

2016-01-01

Human cells respond to DNA damage by elevating sphingosine, a bioactive sphingolipid that induces programmed cell death (PCD) in response to various forms of stress, but its regulation and role in the DNA damage response remain obscure. Herein we demonstrate that DNA damage increases sphingosine levels in tumor cells by upregulating alkaline ceramidase 2 (ACER2) and that the upregulation of the ACER2/sphingosine pathway induces PCD in response to DNA damage by increasing the production of reactive oxygen species (ROS). Treatment with the DNA damaging agent doxorubicin increased both ACER2 expression and sphingosine levels in HCT116 cells in a dose-dependent manner. ACER2 overexpression increased sphingosine in HeLa cells whereas knocking down ACER2 inhibited the doxorubicin-induced increase in sphingosine in HCT116 cells, suggesting that DNA damage elevates sphingosine by upregulating ACER2. Knocking down ACER2 inhibited an increase in the apoptotic and necrotic cell population and the cleavage of poly ADP ribose polymerase (PARP) in HCT116 cells in response to doxorubicin as well as doxorubicin-induced release of lactate dehydrogenase (LDH) from these cells. Similar to treatment with doxorubicin, ACER2 overexpression induced an increase in the apoptotic and necrotic cell population and PARP cleavage in HeLa cells and LDH release from cells, suggesting that ACER2 upregulation mediates PCD in response to DNA damage through sphingosine. Mechanistic studies demonstrated that the upregulation of the ACER2/sphingosine pathway induces PCD by increasing ROS levels. Taken together, these results suggest that the ACER2/sphingosine pathway mediates PCD in response to DNA damage through ROS production. PMID:26943039

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

PubMed

Sowmithra, K; Shetty, N J; Jha, S K; Chaubey, R C

2015-12-01

Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Oxidative DNA damage background estimated by a system model of base excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokhansanj, B A; Wilson, III, D M

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

Aflatoxin B₁-Induced Developmental and DNA Damage in Caenorhabditis elegans.

PubMed

Feng, Wei-Hong; Xue, Kathy S; Tang, Lili; Williams, Phillip L; Wang, Jia-Sheng

2016-12-26

Aflatoxin B₁ (AFB₁) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB₁ has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB₁ is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB₁, using Caenorhabditis elegans as a study model. Results found that AFB₁ induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB₁ inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB₁-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans .

Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation

DTIC Science & Technology

2014-09-18

MEASUREMENTS OF DNA DAMAGE AND REPAIR IN BACILLUS ANTHRACIS STERNE SPORES BY UV RADIATION...AFIT-ENP-T-14-S-01 MEASUREMENTS OF DNA DAMAGE AND REPAIR IN BACILLUS ANTHRACIS STERNE SPORES BY UV RADIATION THESIS Presented to the... DAMAGE AND REPAIR IN BACILLUS ANTHRACIS STERNE SPORES BY UV RADIATION Chelsea C. Marcum, BS Approved

Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response.

PubMed

Cazzalini, Ornella; Scovassi, A Ivana; Savio, Monica; Stivala, Lucia A; Prosperi, Ennio

2010-01-01

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response. 2010 Elsevier B.V. All rights reserved.

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

PubMed

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Kinetic Basis of Nucleotide Selection Employed by a Protein Template-Dependent DNA Polymerase†

PubMed Central

Brown, Jessica A.; Fowler, Jason D.; Suo, Zucai

2010-01-01

Rev1, a Y-family DNA polymerase, contributes to spontaneous and DNA damage-induced mutagenic events. In this paper, we have employed pre-steady state kinetic methodology to establish a kinetic basis for nucleotide selection by human Rev1, a unique nucleotidyl transferase that uses a protein template-directed mechanism to preferentially instruct dCTP incorporation. This work demonstrated that the high incorporation efficiency of dCTP is dependent on both substrates: an incoming dCTP and a templating base dG. The extremely low base substitution fidelity of human Rev1 (100 to 10-5) was due to the preferred misincorporation of dCTP with templating bases dA, dT, and dC over correct dNTPs. Using non-natural nucleotide analogs, we showed that hydrogen bonding interactions between residue R357 of human Rev1 and an incoming dNTP are not essential for DNA synthesis. Lastly, human Rev1 discriminates between ribonucleotides and deoxyribonucleotides mainly by reducing the rate of incorporation, and the sugar selectivity of human Rev1 is sensitive to both the size and orientation of the 2′-substituent of a ribonucleotide. PMID:20518555

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

PubMed

Ogara, María F; Sirkin, Pablo F; Carcagno, Abel L; Marazita, Mariela C; Sonzogni, Silvina V; Ceruti, Julieta M; Cánepa, Eduardo T

2013-01-01

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

PubMed Central

Ganapathy, Vengatesh; Manyanga, Jimmy; Brame, Lacy; McGuire, Dehra; Sadhasivam, Balaji; Floyd, Evan; Rubenstein, David A.; Ramachandran, Ilangovan; Wagener, Theodore

2017-01-01

Background Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. Objective The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. Methods Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. Results EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. Conclusions Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public. PMID:28542301

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.

PubMed

Perucca, Paola; Mocchi, Roberto; Guardamagna, Isabella; Bassi, Elisabetta; Sommatis, Sabrina; Nardo, Tiziana; Prosperi, Ennio; Stivala, Lucia Anna; Cazzalini, Ornella

2018-06-01

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2 Wt and DDB2 PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2 PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity. Copyright © 2018 Elsevier B.V. All rights reserved.

The effects of male age on sperm DNA damage in healthy non-smokers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmid, T; Eskenazi, B; Baumgartner, A

The trend for men to have children at older ages raises concerns that advancing age may increase the production of genetically defective sperm, increasing the risks of transmitting germ-line mutations. We investigated the associations between male age and sperm DNA damage and the influence of several lifestyle factors in a healthy non-clinical group of 80 non-smokers (age: 22-80) with no known fertility problems using the sperm Comet analyses. The average percent of DNA that migrated out of the sperm nucleus under alkaline electrophoresis increased with age (0.18% per year, p=0.006); but there was no age association for damage measured undermore » neutral conditions (p=0.7). Men who consumed >3 cups coffee per day had {approx}20% higher % tail DNA under neutral but not alkaline conditions compared to men who consumed no caffeine (p=0.005). Our findings indicate that (a) older men have increased sperm DNA damage associated with alkali-labile sites or single-strand DNA breaks, and (b) independent of age, men with substantial daily caffeine consumption have increased sperm DNA damage associated with double-strand DNA breaks. DNA damage in sperm can be converted to chromosomal aberrations and gene mutations after fertilization increasing the risks for developmental defects and genetic diseases among offspring.« less

Organic honey supplementation reverses pesticide-induced genotoxicity by modulating DNA damage response.

PubMed

Alleva, Renata; Manzella, Nicola; Gaetani, Simona; Ciarapica, Veronica; Bracci, Massimo; Caboni, Maria Fiorenza; Pasini, Federica; Monaco, Federica; Amati, Monica; Borghi, Battista; Tomasetti, Marco

2016-10-01

Glyphosate (GLY) and organophosphorus insecticides such as chlorpyrifos (CPF) may cause DNA damage and cancer in exposed individuals through mitochondrial dysfunction. Polyphenols ubiquitously present in fruits and vegetables, have been viewed as antioxidant molecules, but also influence mitochondrial homeostasis. Here, honey containing polyphenol compounds was evaluated for its potential protective effect on pesticide-induced genotoxicity. Honey extracts from four floral organic sources were evaluated for their polyphenol content, antioxidant activity, and potential protective effects on pesticide-related mitochondrial destabilization, reactive oxygen and nitrogen species formation, and DNA damage response in human bronchial epithelial and neuronal cells. The protective effect of honey was, then evaluated in a residential population chronically exposed to pesticides. The four honey types showed a different polyphenol profile associated with a different antioxidant power. The pesticide-induced mitochondrial dysfunction parallels ROS formation from mitochondria (mtROS) and consequent DNA damage. Honey extracts efficiently inhibited pesticide-induced mtROS formation, and reduced DNA damage by upregulation of DNA repair through NFR2. Honey supplementation enhanced DNA repair activity in a residential population chronically exposed to pesticides, which resulted in a marked reduction of pesticide-induced DNA lesions. These results provide new insight regarding the effect of honey containing polyphenols on pesticide-induced DNA damage response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Blocking 1800 MHz mobile phone radiation-induced reactive oxygen species production and DNA damage in lens epithelial cells by noise magnetic fields].

PubMed

Wu, Wei; Yao, Ke; Wang, Kai-jun; Lu, De-qiang; He, Ji-liang; Xu, Li-hong; Sun, Wen-jun

2008-01-01

To investigate whether the exposure to the electromagnetic noise can block reactive oxygen species (ROS) production and DNA damage of lens epithelial cells induced by 1800 MHz mobile phone radiation. The DCFH-DA method and comet assay were used respectively to detect the intracellular ROS and DNA damage of cultured human lens epithelial cells induced by 4 W/kg 1800 MHz mobile phone radiation or/and 2 muT electromagnetic noise for 24 h intermittently. 1800 MHz mobile phone radiation at 4 W/kg for 24 h increased intracellular ROS and DNA damage significantly (P<0.05). However, the ROS level and DNA damage of mobile phone radiation plus noise group were not significant enhanced (P>0.05) as compared to sham exposure group. Electromagnetic noise can block intracellular ROS production and DNA damage of human lens epithelial cells induced by 1800 MHz mobile phone radiation.

DNA damage and repair in plants under ultraviolet and ionizing radiations.

PubMed

Gill, Sarvajeet S; Anjum, Naser A; Gill, Ritu; Jha, Manoranjan; Tuteja, Narendra

2015-01-01

Being sessile, plants are continuously exposed to DNA-damaging agents present in the environment such as ultraviolet (UV) and ionizing radiations (IR). Sunlight acts as an energy source for photosynthetic plants; hence, avoidance of UV radiations (namely, UV-A, 315-400 nm; UV-B, 280-315 nm; and UV-C, <280 nm) is unpreventable. DNA in particular strongly absorbs UV-B; therefore, it is the most important target for UV-B induced damage. On the other hand, IR causes water radiolysis, which generates highly reactive hydroxyl radicals (OH(•)) and causes radiogenic damage to important cellular components. However, to maintain genomic integrity under UV/IR exposure, plants make use of several DNA repair mechanisms. In the light of recent breakthrough, the current minireview (a) introduces UV/IR and overviews UV/IR-mediated DNA damage products and (b) critically discusses the biochemistry and genetics of major pathways responsible for the repair of UV/IR-accrued DNA damage. The outcome of the discussion may be helpful in devising future research in the current context.

GSTM1 and GSTT1 Genes are Associated With DNA Damage of p53 Gene in Coke-oven Workers.

PubMed

He, Yuefeng; Qi, Jun; He, Fang; Zhang, Yongchang; Wang, Youlian; Zhang, Ruobing; Li, Gang

2017-06-01

This study investigated whether variations in GSTT1 and GSTM1 gene are associated with the DNA damage level of p53 gene. We quantified urinary 1-hydroxypyrene using high-performance liquid chromatography, and examined the DNA damage level of p53 gene by real-time quantitative PCR in 756 coke-oven workers. Multiplex PCR was used to detect the presence or absence of genes. DNA damage levels of p53 gene in the high exposure group and intermediate exposure group were significantly higher than that of p53 gene in the low exposure group (P < 0.01). In coke-oven workers, the DNA damage levels of subjects with non-null genotype in GSTT1 or GSTM1 gene were significantly higher than that of those with the null genotype (P < 0.01). GSTT1 and GSTM1 may modulate DNA damage levels of p53 gene when exposed to polycyclic aromatic hydrocarbons.

Leucocytes DNA damage in mice exposed to JS-118 by the comet assay.

PubMed

Zhang, Tao; Hu, Jiye; Zhang, Yuchao; Zhao, Qianfei; Ning, Jun

2011-09-01

JS-118 is an extensively used insecticide in China. The present study investigated the genotoxic effect of JS-118 on whole blood at 24, 48, 72 and 96 h by using alkaline comet assay. Male Kunming mice were given 6.25, 12.5, 25, 50 and 100 mg/kg BW of JS-118 intraperitoneally. A statistically significant increase in all comet parameters indicating DNA damage was observed at 24 h post-treatment (p < 0.05). A clear concentration-dependent increase of DNA damage was revealed as evident by the OTM (arbitrary units), tail length (µm) and tail DNA (%). From 48 h post-treatment, a gradual decrease in mean comet parameters was noted. By 96 h of post-treatment, the mean comet tail length reached control levels indicating repair of damaged DNA. This study on mice showed different DNA damage depending on the concentration of JS-118 and the period of treatment. The present study provided further information of the potential risk of the genetic damage caused by JS-118.

Chronic inflammation-related DNA damage response: a driving force of gastric cardia carcinogenesis

PubMed Central

Guo, Yi; Tian, Dongping; Yun, Hailong; Chen, Donglin; Su, Min

2015-01-01

Gastric cardia cancer (GCC) is a highly aggressive disease associated with chronic inflammation. To investigate the relationship between DNA damage response (DDR) and chronic inflammation, we collected 100 non-tumor gastric cardia specimens of Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. A significantly higher proportion of severe chronic inflammation was found in dysplastic epithelia (80.9%) in comparison with that in non-dysplastic tissues (40.7%) (P<0.001). Immunohistochemical analysis demonstrated that DNA damage response was parallel with the chronic inflammation degrees from normal to severe inflammation (P<0.05). We found that DNA damage response was progressively increased with the progression of precancerous lesions (P<0.05). These findings provide pathological evidence that persistent chronic inflammation-related DNA damage response may be a driving force of gastric cardia carcinogenesis. Based on these findings, DNA damage response in non-malignant tissues may become a promising biomedical marker for predicting malignant transformation in the gastric cardia. PMID:25650663

Chronic inflammation-related DNA damage response: a driving force of gastric cardia carcinogenesis.

PubMed

Lin, Runhua; Xiao, Dejun; Guo, Yi; Tian, Dongping; Yun, Hailong; Chen, Donglin; Su, Min

2015-02-20

Gastric cardia cancer (GCC) is a highly aggressive disease associated with chronic inflammation. To investigate the relationship between DNA damage response (DDR) and chronic inflammation, we collected 100 non-tumor gastric cardia specimens of Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. A significantly higher proportion of severe chronic inflammation was found in dysplastic epithelia (80.9%) in comparison with that in non-dysplastic tissues (40.7%) (P<0.001). Immunohistochemical analysis demonstrated that DNA damage response was parallel with the chronic inflammation degrees from normal to severe inflammation (P<0.05). We found that DNA damage response was progressively increased with the progression of precancerous lesions (P<0.05). These findings provide pathological evidence that persistent chronic inflammation-related DNA damage response may be a driving force of gastric cardia carcinogenesis. Based on these findings, DNA damage response in non-malignant tissues may become a promising biomedical marker for predicting malignant transformation in the gastric cardia.

DNA Damage Responses in Prokaryotes: Regulating Gene Expression, Modulating Growth Patterns, and Manipulating Replication Forks

PubMed Central

Kreuzer, Kenneth N.

2013-01-01

Recent advances in the area of bacterial DNA damage responses are reviewed here. The SOS pathway is still the major paradigm of bacterial DNA damage response, and recent studies have clarified the mechanisms of SOS induction and key physiological roles of SOS including a very major role in genetic exchange and variation. When considering diverse bacteria, it is clear that SOS is not a uniform pathway with one purpose, but rather a platform that has evolved for differing functions in different bacteria. Relating in part to the SOS response, the field has uncovered multiple apparent cell-cycle checkpoints that assist cell survival after DNA damage and remarkable pathways that induce programmed cell death in bacteria. Bacterial DNA damage responses are also much broader than SOS, and several important examples of LexA-independent regulation will be reviewed. Finally, some recent advances that relate to the replication and repair of damaged DNA will be summarized. PMID:24097899

Dynamic maps of UV damage formation and repair for the human genome

PubMed Central

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-01-01

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS–Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS–Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage. PMID:28607063

Dynamic maps of UV damage formation and repair for the human genome.

PubMed

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-06-27

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

The Molecular Origin of the MMR-dependent Apoptosis Pathway from Dynamics Analysis of MutSα-DNA Complexes

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R.

2012-01-01

The cellular response to DNA damage signaling by MMR proteins is incompletely understood. It is generally accepted that MMR-dependent apoptosis pathway in response to DNA damage detection is independent of MMR's DNA repair function. In this study we investigate correlated motions in response to the binding of mismatched and PCL DNA fragments by MutSα, as derived from 50 ns molecular dynamics simulations. The protein dynamics in response to the mismatched and damaged DNA recognition suggests that MutSα signals their recognition through independent pathways providing evidence for the molecular origin of the MMR-dependent apoptosis. MSH2 subunit is indicated to play a key role in signaling both mismatched and damaged DNA recognition; localized and collective motions within the protein allow identifying sites on the MSH2 surface possible involved in recruiting proteins responsible for downstream events. Unlike in the mismatch complex, predicted key communication sites specific for the damage recognition are on the list of known cancer causing mutations or deletions. This confirms MSH2's role in signaling DNA-damage induced apoptosis and suggests that defects in MMR alone is sufficient to trigger tumorigenesis, supporting the experimental evidence that MMR-damage response function could protect from the early occurrence of tumors. Identifying these particular communication sites may have implications for the treatment of cancers that are not defective for MMR, but are unable to function optimally for MMR-dependent responses following DNA damage such as the case of resistance to cisplatin. PMID:22712459

Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muniz, Juan F.; McCauley, Linda; Scherer, J.

Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers andmore » applicators (p < 0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects.« less

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

Study of the effect of simulated space environment on nucleoprotein and DNA thin films

NASA Astrophysics Data System (ADS)

Fekete, A.; Módos, K.; Hegedüs, M.; Rontó, Gy.; Kovács, G.; Bérces, A.; Kargl, G.; Kömle, N. I.; Lammer, H.

2002-11-01

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of life is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (λ = 254 nm) and high vacuum (10-5 mbar). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA.

Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

NASA Astrophysics Data System (ADS)

Fekete, A.; Rontó, Gy.; Hegedüs, M.; Módos, K.; Bérces, A.; Kovács, G.; Lammer, H.; Panitz, C.

2004-01-01

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation ( λ=254 nm) and high vacuum (10 -4 Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA.

Effects of different levels of vitamin C on UV radiation-induced DNA damage

NASA Astrophysics Data System (ADS)

Zhou, Dianfeng; Heng, Hang; Ji, Kang; Ke, Weizhong

2005-05-01

The Raman spectra of DNA in different levels of vitamin C with 10- and 30-min ultraviolet (UV) radiations were reported. The intensity of UV radiation was 18.68 W/m2. The experimental results proved that vitamin C could alone prevent UV radiation from damaging DNA, but the effects depended on the concentration of vitamin C. When the concentration of vitamin C was about 0.08-0.4 mmol/L, vitamin C decreased UV radiation-induced DNA's damage. When the concentration of vitamin C exceeded 0.4 mmol/L, vitamin C accelerated DNA's damage instead. Maybe the reason is that when DNA in aqueous solution is radiated by UV, free radicals come into being, and vitamin C can scavenge free radicals, so vitamin C in lower concentration can protect DNA. The quantity of free radicals is finite, when vitamin C is superfluous, free radicals have been scavenged absolutely and vitamin C is residual. Vitamin C is a strong reductant. When the mixture of DNA and residual vitamin C is radiated by UV, vitamin C reacts with DNA. The more residual vitamin C and the longer time of UV radiation, the more DNA is damaged.

Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator of hydrogen peroxide in human fibroblasts.

PubMed

Salazar, J J; Van Houten, B

1997-11-01

To test the hypothesis that mitochondrial DNA (mtDNA) is more prone to reactive oxygen species (ROS) damage than nuclear DNA, a continuous flux of hydrogen peroxide (H2O2) was produced with the glucose/glucose oxidase system. Using a horse radish peroxidase (HRPO)-based colorimetric assay to detect H2O2, glucose oxidase (GO; 12 mU/ml) produced 95 microM of H2O2 in 1 h, whereas only 46 microM of hydrogen peroxide accumulated in the presence of SV40-transformed human fibroblasts ( approximately 1 x 10(6). DNA damage was assessed in the mitochondira and three nuclear regions using a quantitative PCR assay. GO (12 mU/ml) resulted in more damage to the mitochondrial DNA (2.250 +/- 0.045 lesions/10 kb) than in any one of three nuclear targets, which included the non-expressed beta-globin locus (0.436 +/- 0.029 lesions/10 kb); and the active DNA polymerase b gene (0.442 +/- 0.037 lesions/10 kb); and the active hprt gene (0.310 +/- 0.025). Damage to the mtDNA occurred within 15 min of GO treatment, whereas nuclear damage did not appear until after 30 min, and reached a maximum after 60 min. Repair of mitochondrial damage after a 15 min GO (6 mU/ml) treatment was examined. Mitochondria repaired 50% of the damage after 1 h, and by 6 h all the damage was repaired. Higher doses of GO-generated H202, or more extended treatment periods, lead to mitochondrial DNA damage which was not repaired. Mitochondrial function was monitored using the MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. A 15 min treatment with 6 mU/ml of GO decreased mitochondrial activity to 80% of the control; the activity recovered completely within 1 h after damage. These data show that GO-generated H202 causes acute damage to mtDNA and function, and demonstrate that this organelle is an important site for the cellular toxicity of ROS.

Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

PubMed

Douthwright, Stephen; Sluder, Greenfield

2014-10-01

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. © 2014 Wiley Periodicals, Inc.

Is there a similarity between DNA damage in adults with chronic alcoholism and community-dwelling healthy older adults?

PubMed

Retana-Ugalde, Raquel; Altamirano-Lozano, Mario; Mendoza-Núñez, Víctor Manuel

2007-01-01

Daily alcohol consumption and ageing have been linked with DNA damage, leading to the hypothesis that chronic alcoholism causes DNA damage similar to that which occurs with ageing. Likewise, it has been suggested that chronic alcoholism is the cause of accelerated or premature ageing. The objective of this study was to evaluate the frequency and magnitude of DNA damage among adults with chronic alcoholism and healthy older adults residing in Mexico City. A cross-sectional and comparative study was carried out in a sample of 53 chronic alcoholics of 25-44 years of age (without alcohol ingestion in the past 30 days) without additional diseases, 26 healthy subjects >or=60 years of age, and 25 healthy adults of 25-44 years of age without alcohol addiction, all residents of Mexico City during the past 10 years. DNA damage was evaluated by single-cell gel electrophoresis technique (Comet assay). Our results showed a similar percentage of DNA damage between healthy elderly subjects and chronic alcoholics (62 vs 55%, P >0.05), although average DNA migration was greater in alcoholics than in the elderly (78.1 +/- 33.2 vs 58.6 +/- 26.2, P = 0.09). However, the percentage of subjects with more than six damaged cells was higher in the older adults subjects group than in the group chronic alcoholics (19 vs 35%, P = 0.16). Data suggest that DNA damage is not similar in young subjects with chronic alcoholism that which occurs with ageing.

Chk1 Promotes DNA Damage Response Bypass following Oxidative Stress in a Model of Hydrogen Peroxide-Associated Ulcerative Colitis through JNK Inactivation and Chromatin Binding

PubMed Central

Silver, Andrew; Guenther, Thomas; Siedentopf, Sandra; Ross, Jochen; Vo, Diep-Khanh; Roessner, Albert

2017-01-01

Dysregulation of c-Jun N-terminal kinase (JNK) activation promoted DNA damage response bypass and tumorigenesis in our model of hydrogen peroxide-associated ulcerative colitis (UC) and in patients with quiescent UC (QUC), UC-related dysplasia, and UC-related carcinoma (UC-CRC), thereby adapting to oxidative stress. In the UC model, we have observed features of oncogenic transformation: increased proliferation, undetected DNA damage, and apoptosis resistance. Here, we show that Chk1 was downregulated but activated in the acute and quiescent chronic phases. In both phases, Chk1 was linked to DNA damage response bypass by suppressing JNK activation following oxidative stress, promoting cell cycle progression despite DNA damage. Simultaneously, activated Chk1 was bound to chromatin. This triggered histone acetylation and the binding of histone acetyltransferases and transcription factors to chromatin. Thus, chromatin-immobilized activated Chk1 executed a dual function by suppressing DNA damage response and simultaneously inducing chromatin modulation. This caused undetected DNA damage and increased cellular proliferation through failure to transmit the appropriate DNA damage signal. Findings in vitro were corroborated by chromatin accumulation of activated Chk1, Ac-H3, Ac-H4, and c-Jun in active UC (AUC) in vivo. Targeting chromatin-bound Chk1, GCN5, PCAF, and p300/CBP could be a novel therapeutic strategy to prevent UC-related tumor progression. PMID:28751935

Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes

PubMed Central

Pieper, Andrew A.; Brat, Daniel J.; Krug, David K.; Watkins, Crystal C.; Gupta, Alok; Blackshaw, Seth; Verma, Ajay; Wang, Zhao-Qi; Snyder, Solomon H.

1999-01-01

Streptozotocin (STZ) selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme whose overactivation by DNA strand breaks depletes its substrate NAD+ and then ATP, leading to cellular death from energy depletion. We demonstrate DNA damage and a major activation of PARP in pancreatic islets of STZ-treated mice. These mice display a 500% increase in blood glucose and major pancreatic islet damage. In mice with homozygous targeted deletion of PARP (PARP −/−), blood glucose and pancreatic islet structure are normal, indicating virtually total protection from STZ diabetes. Partial protection occurs in PARP +/− animals. Thus, PARP activation may participate in the pathophysiology of type I diabetes, for which PARP inhibitors might afford therapeutic benefit. PMID:10077636

Oxidative DNA damage induced by a hydroperoxide derivative of cyclophosphamide.

PubMed

Murata, Mariko; Suzuki, Toshinari; Midorikawa, Kaoru; Oikawa, Shinji; Kawanishi, Shosuke

2004-09-15

Interstrand DNA cross-linking has been considered to be the primary action mechanism of cyclophosphamide (CP) and its hydroperoxide derivative, 4-hydroperoxycyclophosphamide (4-HC). To clarify the mechanism of anti-tumor effects by 4-HC, we investigated DNA damage in a human leukemia cell line, HL-60, and its H(2)O(2)-resistant clone HP100. Apoptosis DNA ladder formation was detected in HL-60 cells treated with 4-HC, whereas it was not observed in HP100 cells. 4-HC significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, a marker of oxidative DNA damage, in HL-60 cells. On the other hand, CP did not significantly induce 8-oxodG formation and apoptosis in HL-60 cells under the same conditions as did 4-HC. Using (32)P-labeled DNA fragments from the human p53 tumor suppressor gene, 4-HC was found to cause Cu(II)-mediated oxidative DNA damage, but CP did not. Catalase inhibited 4-HC-induced DNA damage, including 8-oxodG formation, suggesting the involvement of H(2)O(2). The generation of H(2)O(2) during 4-HC degradation was ascertained by procedures using scopoletin and potassium iodide. We conclude that, in addition to DNA cross-linking, oxidative DNA damage through H(2)O(2) generation may participate in the anti-tumor effects of 4-HC.

Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

PubMed

Schons-Fonseca, Luciane; da Silva, Josefa B; Milanez, Juliana S; Domingos, Renan H; Smith, Janet L; Nakaya, Helder I; Grossman, Alan D; Ho, Paulo L; da Costa, Renata M A

2016-02-18

We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

PubMed Central

Nair, Nidhi; Shoaib, Muhammad

2017-01-01

Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage. PMID:28698521

APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

PubMed

Local, Andrea; Zhang, Hongying; Benbatoul, Khalid D; Folger, Peter; Sheng, Xia; Tsai, Cheng-Yu; Howell, Stephen B; Rice, William G

2018-06-01

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G 0 -G 1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253) 3 ]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253) 3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253) 3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR . ©2018 American Association for Cancer Research.

Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages.

PubMed

Cannan, Wendy J; Tsang, Betty P; Wallace, Susan S; Pederson, David S

2014-07-18

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages*

PubMed Central

Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.

2014-01-01

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506

Requirement of the Saccharomyces cerevisiae APN1 Gene for the Repair of Mitochondrial DNA Alkylation Damage

PubMed Central

Acevedo-Torres, Karina; Fonseca-Williams, Sharon; Ayala-Torres, Sylvette; Torres-Ramos, Carlos A.

2010-01-01

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. PMID:19197988

Impact of genomic damage and ageing on stem cell function

PubMed Central

Behrens, Axel; van Deursen, Jan M.; Rudolph, K. Lenhard; Schumacher, Björn

2014-01-01

Impairment of stem cell function contributes to the progressive deterioration of tissue maintenance and repair with ageing. Evidence is mounting that age-dependent accumulation of DNA damage in both stem cells and cells that comprise the stem cell microenvironment are partly responsible for stem cell dysfunction with ageing. Here, we review the impact of the various types of DNA damage that accumulate with ageing on stem cell functionality, as well as the development of cancer. We discuss DNA-damage-induced cell intrinsic and extrinsic alterations that influence these processes, and review recent advances in understanding systemic adjustments to DNA damage and how they affect stem cells. PMID:24576896

Repair Mechanism of UV-damaged DNA in Xeroderma Pigmentosum | Center for Cancer Research

Cancer.gov

Xeroderma pigmentosum (XP) is a rare, inherited disorder characterized by extreme skin sensitivity to ultraviolet (UV) rays from sunlight. XP is caused by mutations in genes involved in nucleotide excision repair (NER) of damaged DNA. Normal cells are usually able to fix this damage before it leads to problems; however, the DNA damage is not repaired normally in patients with

Comet assay: a prognostic tool for DNA integrity assessment in infertile men opting for assisted reproduction.

PubMed

Shamsi, M B; Venkatesh, S; Tanwar, M; Singh, G; Mukherjee, S; Malhotra, N; Kumar, R; Gupta, N P; Mittal, S; Dada, R

2010-05-01

The growing concern on transmission of genetic diseases in assisted reproduction technique (ART) and the lacunae in the conventional semen analysis to accurately predict the semen quality has led to the need for new techniques to identify the best quality sperm that can be used in assisted procreation techniques. This study analyzes the sperm parameters in the context of DNA damage in cytogenetically normal, AZF non deleted infertile men for DNA damage by comet assay. Seventy infertile men and 40 fertile controls were evaluated for the semen quality by conventional semen parameters and the sperms were also analyzed for DNA integrity by comet assay. The patients were classified into oligozoospermic (O), asthenozoospermic (A), teratozoospermic (T), oligoasthenoteratozoospermic (OAT) categories and infertile men with normal semen profile. The extent of DNA damage was assessed by visual scoring method of comets. Idiopathic infertile men with normal semen profile (n=18) according to conventional method and patients with history of spontaneous abortions and normal semen profile (n=10) had high degree of DNA damage (29 and 47% respectively) as compared to fertile controls (7%). The O, A, T and OAT categories of patients had a variably higher DNA damage load as compared to fertile controls. The normal range and threshold for DNA damage as a predictor of male fertility potential and technique which could assess the sperm DNA damage are necessary to lower the trauma of couples experiencing recurrent spontaneous abortion or failure in ART.

Increased levels of mitochondrial DNA copy number in patients with vitiligo.

PubMed

Vaseghi, H; Houshmand, M; Jadali, Z

2017-10-01

Oxidative stress is known to be involved in the pathogenesis of autoimmune diseases such as vitiligo. Evidence suggests that the human mitochondrial DNA copy number (mtDNAcn) is vulnerable to damage mediated by oxidative stress. The purpose of this study was to examine and compare peripheral blood mtDNAcn and oxidative DNA damage byproducts (8-hydroxy-2-deoxyguanosine; 8-OHdG) in patients with vitiligo and healthy controls (HCs). The relative mtDNAcn and the oxidative damage (formation of 8-OHdG in mtDNA) of each sample were determined by real-time quantitative PCR. Blood samples were obtained from 56 patients with vitiligo and 46 HCs. The mean mtDNAcn and the degree of mtDNA damage were higher in patients with vitiligo than in HCs. These data suggest that increase in mtDNAcn and oxidative DNA damage may be involved in the pathogenesis of vitiligo. © 2017 British Association of Dermatologists.

A core hSSB1–INTS complex participates in the DNA damage response

PubMed Central

Zhang, Feng; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Human single-stranded DNA-binding protein 1 (hSSB1) plays an important role in the DNA damage response and the maintenance of genomic stability. It has been shown that the core hSSB1 complex contains hSSB1, INTS3 and C9orf80. Using protein affinity purification, we have identified integrator complex subunit 6 (INTS6) as a major subunit of the core hSSB1 complex. INTS6 forms a stable complex with INTS3 and hSSB1 both in vitro and in vivo. In this complex, INTS6 directly interacts with INTS3. In response to the DNA damage response, along with INTS3 and hSSB1, INTS6 relocates to the DNA damage sites. Moreover, the hSSB1–INTS complex regulates the accumulation of RAD51 and BRCA1 at DNA damage sites and the correlated homologous recombination. PMID:23986477

Ex vivo study for the assessment of behavioral factor and gene polymorphisms in individual susceptibility to oxidative DNA damage metals-induced.

PubMed

Di Pietro, Angela; Baluce, Barbara; Visalli, Giuseppa; La Maestra, Sebastiano; Micale, Rosanna; Izzotti, Alberto

2011-06-01

Transition metals in fine particulate matter generated by combustion induce oxidative DNA damage and inflammation. However, there is remarkable inter-individual variability in susceptibility to these damages. To assess this variability, an ex vivo study was performed using lymphocytes of 47 Caucasian healthy subjects. Cell samples were exposed to a water solution of oil fly ash (OFA). This was formed by the distinctive transition metals vanadium, iron, and nickel. Oxidative DNA damage was evaluated by testing cell viability, intracellular ROS production and 8-oxo-dG. DNA fragmentation and DNA repair capacity were assessed by using the Alkaline-Halo assay. GSTM1, GSTT1, hOGG1, and C677T and A1298C MTHFR gene polymorphisms were tested. Demographic and behavioral factors, collected by questionnaire, were also considered. OFA induced damages showed: (a) a 20-fold variation in range among different subjects in ROS production, (b) a 7-fold variation in range of 8-oxo-dG, and (c) a 25-fold variation in range in DNA repair capacity. A significant increase in DNA damage was detected in GSTT1-deficent subjects compared with wild type genotype carriers. Increases in cytoplasmic ROS and decreases in DNA repair capacity (P<0.05) were observed in C677T and A1298C variants of MTHFR. A remarkable protective effect of high fruits and vegetable intake was observed for ROS production and DNA damage. Conversely, an adverse effect of meat intake was observed on ROS increase, DNA damage and repair capacity, probably due to the increased intake of bioavailable iron. Smoking decreased DNA repair capacity, while age increased OFA-induced DNA damage. The wide comparative analysis of the complex interactions network, between genetic and behavioral factors provides evidence of the remarkable role of several lifestyle factors. In comparison to genetic polymorphisms they seem to have a higher weight in determining individual susceptibility to the adverse effects of airborne pollutants as transition metals. Copyright © 2011 Elsevier GmbH. All rights reserved.

Origins and consequences of DNA damage in male germ cells.

PubMed

Aitken, R John; De Iuliis, Geoffry N

2007-06-01

DNA damage in the male germline is associated with poor fertilization rates following IVF, defective preimplantation embryonic development, and high rates of miscarriage and morbidity in the offspring, including childhood cancer. This damage is poorly characterized, but is known to involve hypomethylation of key genes, oxidative base damage, endonuclease-mediated cleavage and the formation of adducts with xenobiotics and the products of lipid peroxidation. There are many possible causes of such DNA damage, including abortive apoptosis, the oxidative stress associated with male genital tract infection, exposure to redox cycling chemicals, and defects of spermiogenesis associated with the retention of excess residual cytoplasm. Physical factors such as exposure to radiofrequency electromagnetic radiation or mild scrotal heating can also induce DNA damage in mammalian spermatozoa, although the underlying mechanisms are unclear. Ultimately, resolving the precise nature of the DNA lesions present in the spermatozoa of infertile men will be an important step towards uncovering the aetiology of this damage and developing strategies for its clinical management.

High basal metabolic rate does not elevate oxidative stress during reproduction in laboratory mice.

PubMed

Brzęk, Paweł; Książek, Aneta; Ołdakowski, Łukasz; Konarzewski, Marek

2014-05-01

Increased oxidative stress (OS) has been suggested as a physiological cost of reproduction. However, previous studies reported ambiguous results, with some even showing a reduction of oxidative damage during reproduction. We tested whether the link between reproduction and OS is mediated by basal metabolic rate (BMR), which has been hypothesized to affect both the rate of radical oxygen species production and antioxidative capacity. We studied the effect of reproduction on OS in females of laboratory mice divergently selected for high (H-BMR) and low (L-BMR) BMR, previously shown to differ with respect to parental investment. Non-reproducing L-BMR females showed higher oxidative damage to lipids (quantified as the level of malondialdehyde in internal organ tissues) and DNA (quantified as the level of 8-oxodG in blood serum) than H-BMR females. Reproduction did not affect oxidative damage to lipids in either line; however, it reduced damage to DNA in L-BMR females. Reproduction increased catalase activity in liver (significantly stronger in L-BMR females) and decreased it in kidneys. We conclude that the effect of reproduction on OS depends on the initial variation in BMR and varies between studied internal organs and markers of OS.

Reactive oxygen-mediated damage to a human DNA replication and repair protein.

PubMed

Montaner, Beatriz; O'Donovan, Peter; Reelfs, Olivier; Perrett, Conal M; Zhang, Xiaohong; Xu, Yao-Zhong; Ren, Xiaolin; Macpherson, Peter; Frith, David; Karran, Peter

2007-11-01

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.

Beyond xeroderma pigmentosum: DNA damage and repair in an ecological context. A tribute to James E. Cleaver.

PubMed

Karentz, Deneb

2015-01-01

The ability to repair DNA is a ubiquitous characteristic of life on Earth and all organisms possess similar mechanisms for dealing with DNA damage, an indication of a very early evolutionary origin for repair processes. James E. Cleaver's career (initiated in the early 1960s) has been devoted to the study of mammalian ultraviolet radiation (UVR) photobiology, specifically the molecular genetics of xeroderma pigmentosum and other human diseases caused by defects in DNA damage recognition and repair. This work by Jim and others has influenced the study of DNA damage and repair in a variety of taxa. Today, the field of DNA repair is enhancing our understanding of not only how to treat and prevent human disease, but is providing insights on the evolutionary history of life on Earth and how natural populations are coping with UVR-induced DNA damage from anthropogenic changes in the environment such as ozone depletion. © 2014 The American Society of Photobiology.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

The Causal Relationship between DNA Damage Induction in Bovine Lymphocytes and the Fukushima Nuclear Power Plant Accident

PubMed Central

Nakamura, Asako J.; Suzuki, Masatoshi; Redon, Christophe E.; Kuwahara, Yoshikazu; Yamashiro, Hideaki; Abe, Yasuyuki; Takahashi, Shintaro; Fukuda, Tomokazu; Isogai, Emiko; Bonner, William M.; Fukumoto, Manabu

2017-01-01

The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocyto-fluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident. PMID:28240558

The Causal Relationship between DNA Damage Induction in Bovine Lymphocytes and the Fukushima Nuclear Power Plant Accident.

PubMed

Nakamura, Asako J; Suzuki, Masatoshi; Redon, Christophe E; Kuwahara, Yoshikazu; Yamashiro, Hideaki; Abe, Yasuyuki; Takahashi, Shintaro; Fukuda, Tomokazu; Isogai, Emiko; Bonner, William M; Fukumoto, Manabu

2017-05-01

The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocytofluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident.

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

PubMed

Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

1998-10-01

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

[UV-induced DNA damage and protective effects of antioxidants on DNA damage in human lens epithelial cells studied with comet assay].

PubMed

Wu, Zhi-hong; Wang, Mian-rong; Yan, Qi-chang; Pu, Wei; Zhang, Jin-song

2006-11-01

To investigate the mechanism of UV-induced DNA damage and repair and the protective effects of antioxidants on DNA damage in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group), 2.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated group 1 - 4). The amounts of DNA single strand breaks (SSB) were measured with the alkaline comet assay (CA). The spontaneous repair of DNA SSB after exposure to UV at 10.0 mJ/cm(2) was also determined in human lens epithelial cells. Human lens epithelial cells were treated with different concentration of VitaminC (VitC), taurine, superoxide dismutase (SOD) and epigallocatechin gallate (EGCG) before and after ultraviolet radiation, the effects of antioxidants on DNA damage was examined with alkaline comet assay. The amount of DNA SSB in control group and treated groups 1 - 4 showed increased tendency, was dose-dependent to the dose of UV irradiation, the differences of DNA SSB in 5 group were significantly (P < 0.01). UV-induced DNA SSB at 10.0 mJ/cm(2) in human lens epithelial cells, the half repair time was 60 minutes. Human lens epithelial cells were treated with different concentrations of taurine, SOD and EGCG before ultraviolet radiation. The differences of DNA damage in control and various antioxidant treated groups was statistically significant (F = 6.591, 13.542, 4.626 in cells treated with taurine, SOD and EGCG, respectively, P < 0.01), the difference of VitC effect on DNA in control and treated group were not significantly (F = 1.451, P > 0.05). Human lens epithelial cells were treated with different concentration of VitC, taurine, SOD and EGCG after ultraviolet radiation. The differences of DNA damage between the control and treated group were statistically significant (F = 6.571, 4.810, 6.824, 9.182 in cells treated with VitC, taurine, SOD and EGCG, respectively, P < 0.01). The differences of protective effects on DNA damage in these four different kinds of antioxidants added before UV irradiation were statistically significant (P < 0.01). The differences of protective effects on DNA damage in these four different kinds of antioxidant added after UV irradiation were not significantly (P > 0.05). UV irradiation has a dose-dependent effect on the DNA SSB of lens epithelial cells. Exogenesis VitC, taurine, SOD, EGCG possess protective effective to UV-induced DNA damage. SOD is one of the most powerful antioxidants if added before the UV irradiation and followed by EGCG, taurine and VitC orderly. Four kinds of antioxidants show no apparently differences added after UV-irradiation. SOD and EGCG both are powerful antioxidants.

Effect of complex polyphenols and tannins from red wine (WCPT) on chemically induced oxidative DNA damage in the rat.

PubMed

Casalini, C; Lodovici, M; Briani, C; Paganelli, G; Remy, S; Cheynier, V; Dolara, P

1999-08-01

Flavonoids are polyphenolic antioxidants occurring in vegetables and fruits as well as beverages such as tea and wine which have been thought to influence oxidative damage. We wanted to verify whether a complex mixture of wine tannins (wine complex polyphenols and tannins, WCPT) prevent chemically-induced oxidative DNA damage in vivo. Oxidative DNA damage was evaluated by measuring the ratio of 8-hydroxy-2'-deoxyguanosine (80HdG)/ 2-deoxyguanosine (2dG) x 10(-6) in hydrolyzed DNA using HPLC coupled with electrochemical and UV detectors. We treated rats with WCPT (57 mg/kg p.o.) for 14 d, a dose 10-fold higher than what a moderate wine drinker would be exposed to. WCPT administration significantly reduced the ratio of 80HdG/2dG x 10(-6) in liver DNA obtained from rats treated with 2-nitropropane (2NP) relative to controls administered 2NP only (33. 3 +/- 2.5 vs. 44.9 +/- 3.2 x 10(-6) 2dG; micro +/- SE; p<0.05). On the contrary, pretreatment with WCPT for 10 d did not protect the colon mucosa from oxidative DNA damage induced by 1, 2-dimethylhydrazine (DMH). 2NP and DMH are hepatic and colon carcinogens, respectively, capable of inducing oxidative DNA damage. WCPT have protective action against some types of chemically-induced oxidative DNA damage in vivo.

Myeloid cell leukaemia 1 has a vital role in retinoic acid-mediated protection of Toll-like receptor 9-stimulated B cells from spontaneous and DNA damage-induced apoptosis.

PubMed

Holm, Kristine L; Indrevaer, Randi L; Myklebust, June Helen; Kolstad, Arne; Moskaug, Jan Øivind; Naderi, Elin H; Blomhoff, Heidi K

2016-09-01

Vitamin A is an essential anti-infective agent with pleiotropic effects on cells of the immune system. The goal of the present study was to unravel the impact of the vitamin A metabolite retinoic acid (RA) on B-cell survival related both to normal B-cell homeostasis and to the detrimental effects imposed by DNA-damaging agents. By combining RA with Toll-like receptor 9 (TLR9) ligands, we show that RA prevents spontaneous, irradiation- and doxorubicin-induced apoptosis of human B cells in an RA receptor-dependent manner. RA-mediated survival involved up-regulation of the anti-apoptotic protein myeloid cell leukemia 1 (MCL1) at the transcriptional level, and knock down of MCL1 by small interfering RNA partially reversed the effects of RA. To ensure that the combination of TLR9-ligands and RA would not promote the survival of malignant B cells, the combined effects of stimulation with RA and TLR9 ligands was assessed on cells from patients with B-cell malignancies. In contrast to the effects on normal B cells, the combination of TLR9 stimulation and RA neither enhanced the MCL1 levels nor inhibited the death of malignant B cells challenged by DNA-damaging agents. Taken together, the present results reveal a vital role of MCL1 in RA-mediated survival of normal B cells. Moreover, the findings suggest that RA in combination with TLR9 ligands might be useful adjuvants in the treatment of B-cell malignancies by selectively protecting normal and not malignant B cells from DNA-damage-induced cell death. © 2016 John Wiley & Sons Ltd.

Radiation damage to nucleoprotein complexes in macromolecular crystallography

DOE PAGES

Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; ...

2015-01-30

Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. Despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under themore » same controlled conditions. A model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N 1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.« less

Identification of evolutionarily conserved DNA damage response genes that alter sensitivity to cisplatin

PubMed Central

Gaponova, Anna V.; Deneka, Alexander Y.; Beck, Tim N.; Liu, Hanqing; Andrianov, Gregory; Nikonova, Anna S.; Nicolas, Emmanuelle; Einarson, Margret B.; Golemis, Erica A.; Serebriiskii, Ilya G.

2017-01-01

Ovarian, head and neck, and other cancers are commonly treated with cisplatin and other DNA damaging cytotoxic agents. Altered DNA damage response (DDR) contributes to resistance of these tumors to chemotherapies, some targeted therapies, and radiation. DDR involves multiple protein complexes and signaling pathways, some of which are evolutionarily ancient and involve protein orthologs conserved from yeast to humans. To identify new regulators of cisplatin-resistance in human tumors, we integrated high throughput and curated datasets describing yeast genes that regulate sensitivity to cisplatin and/or ionizing radiation. Next, we clustered highly validated genes based on chemogenomic profiling, and then mapped orthologs of these genes in expanded genomic networks for multiple metazoans, including humans. This approach identified an enriched candidate set of genes involved in the regulation of resistance to radiation and/or cisplatin in humans. Direct functional assessment of selected candidate genes using RNA interference confirmed their activity in influencing cisplatin resistance, degree of γH2AX focus formation and ATR phosphorylation, in ovarian and head and neck cancer cell lines, suggesting impaired DDR signaling as the driving mechanism. This work enlarges the set of genes that may contribute to chemotherapy resistance and provides a new contextual resource for interpreting next generation sequencing (NGS) genomic profiling of tumors. PMID:27863405

Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response

PubMed Central

Guidi, Riccardo; Guerra, Lina; Levi, Laura; Stenerlöw, Bo; Fox, James G.; Josenhans, Christine; Masucci, Maria G.; Frisan, Teresa

2014-01-01

Summary Epidemiological evidence links chronic bacterial infections to the increased incidence of certain types of cancer but the molecular mechanisms by which bacteria contribute to tumour initiation and progression are still poorly characterized. Here we show that chronic exposure to the genotoxin cytolethal distending toxin (CDT) of Gram-negative bacteria promotes genomic instability and acquisition of phenotypic properties of malignancy in fibroblasts and colon epithelial cells. Cells grown for more than 30 weeks in the presence of sublethal doses of CDT showed increased mutation frequency, and accumulation of chromatin and chromosomal aberrations in the absence of significant alterations of cell cycle distribution, decreased viability or senescence. Cell survival was dependent on sustained activity of the p38 MAP kinase. The ongoing genomic instability was associated with impaired activation of the DNA damage response and failure to efficiently activate cell cycle checkpoints upon exposure to genotoxic stress. Independently selected sublines showed enhanced anchorage-independent growth as assessed by the formation of colonies in semisolid agarose. These findings support the notion that chronic infection by CDT-producing bacteria may promote malignant transformation, and point to the impairment of cellular control mechanisms associated with the detection and repair of DNA damage as critical events in the process. PMID:22998585

Structural basis of detection and signaling of DNA single-strand breaks by human PARP-1

DOE PAGES

Eustermann, Sebastian; Wu, Wing -Fung; Langelier, Marie -France; ...

2015-11-25

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformabilitymore » of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Finally, our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.« less

UVA-potentiated damage to calf thymus DNA by Fenton reaction system and protection by para-aminobenzoic acid.

PubMed

Shih, M K; Hu, M L

1996-03-01

Calf thymus DNA was irradiated with low-intensity UVA (main output at 365 nm, 2 mW cm-2 or 36 kJ m-2 for 30 min), and the role of metal ions, hydrogen peroxide and reactive oxygen species (ROS) was examined. DNA damage was measured as thiobarbituric acid-reactive substances (possibly from degradation of deoxyribose) and as changes in ethidium bromide-DNA fluorescence due to unwinding from strand breaks. Under the present experimental conditions, UVA alone or in the presence of H2O2 had no effect on DNA but slightly enhanced the damage by iron/EDTA. Ultraviolet A strongly enhanced DNA damage (ca four- to five-fold) by the Fenton reaction system (50 microM Fe2+/100 microM EDTA + 0.5 mM H2O2). The results suggest that the Fenton reaction system was "photosensitized" to damage DNA by low-intensity UVA radiation. The enhanced damage by UVA was attributed in part to the reduction of Fe3+ to Fe2+. Ultraviolet A had no effect when iron (ferric or ferrous) ions were replaced by Cu2+, Zn2+, Mn2+ or Cd2+. The ROS involved in the UVA-enhanced damage to DNA by the Fenton reagents were OH and, to a lesser extent, superoxide anions. The UVA-potentiated DNA damage by the Fenton reaction system was then used to examine the protective effect of para-aminobenzoate (PABA), a UVB-absorbing sunscreen that protects against photocarcinogenesis in hairless mice. The results show that PABA and mannitol dose-dependently inhibited the damage with concentrations required for 50% inhibition at 0.1 mM and 3 mM, respectively. The protection by PABA was attributed to its radical-scavenging ability because PABA does not absorb light in the UVA region. These findings may be relevant to the biological damage by UVA and suggest that PABA is useful in protection against photocarcinogenesis by wide-range UV radiation.

Tandem Mass Spectrometry for Characterization of Covalent Adducts of DNA with Anti-cancer Therapeutics

PubMed Central

Silvestri, Catherine; Brodbelt, Jennifer S.

2012-01-01

The chemotherapeutic activities of many anticancer and antibacterial drugs arise from their interactions with nucleic acid substrates. Some of these ligands interact with DNA in a way that causes conformational changes or damage to the nucleic acid targets, ultimately altering recognition by key DNA-specific enzymes, interfering with DNA transcription or prohibiting replication, and terminating cell growth and proliferation. The design and synthesis of ligands that bind to nucleic acids remains a dynamic field in medicinal chemistry and pharmaceutical research. The quest for more selective and efficacious DNA-interactive anti-cancer chemotherapeutics has likewise catalyzed the need for sensitive analytical methods that can provide structural information about the nature of the resulting DNA adducts and provide insight into the mechanistic pathways of the DNA/drug interactions and the impact on the cellular processes in biological systems. This review focuses on the array of tandem mass spectrometric strategies developed and applied for characterization of covalent adducts formed between DNA and anti-cancer ligands. PMID:23150278

Estimation and quantification of human DNA in dental calculus: A pilot study.

PubMed

Singh, Udita; Goel, Saurabh

2017-01-01

Identification using DNA has proved its accuracy multiple times in the field of forensic investigations. Investigators usually rely on either teeth or bone as the DNA reservoirs. However, there are instances where the skeletal or dental remains are not available or not preserved properly. Moreover, due to religious beliefs, the family members of the dead do not allow the investigating team to damage the remains for the sole purpose of identification. To investigate the presence of human DNA in dental calculus and to quantify the amount, if present. This prospective single-blinded pilot study included twenty subjects selected from the patients visiting a dental college. The samples of dental calculus were collected from the thickest portion of calculus deposited on the lingual surfaces of mandibular incisors. These samples were decontaminated and subjected to gel electrophoresis for DNA extraction. DNA was found in 85% cases. The amount of DNA varied from 21 to 37 μg/ml of dental calculus. Dental calculus is a rich reservoir of human DNA.

Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

PubMed

Ceruti, Julieta M; Scassa, María E; Marazita, Mariela C; Carcagno, Abel C; Sirkin, Pablo F; Cánepa, Eduardo T

2009-06-01

p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

Effects of atmospheric pressure plasmas on isolated and cellular DNA-a review.

PubMed

Arjunan, Krishna Priya; Sharma, Virender K; Ptasinska, Sylwia

2015-01-29

Atmospheric Pressure Plasma (APP) is being used widely in a variety of biomedical applications. Extensive research in the field of plasma medicine has shown the induction of DNA damage by APP in a dose-dependent manner in both prokaryotic and eukaryotic systems. Recent evidence suggests that APP-induced DNA damage shows potential benefits in many applications, such as sterilization and cancer therapy. However, in several other applications, such as wound healing and dentistry, DNA damage can be detrimental. This review reports on the extensive investigations devoted to APP interactions with DNA, with an emphasis on the critical role of reactive species in plasma-induced damage to DNA. The review consists of three main sections dedicated to fundamental knowledge of the interactions of reactive oxygen species (ROS)/reactive nitrogen species (RNS) with DNA and its components, as well as the effects of APP on isolated and cellular DNA in prokaryotes and eukaryotes.

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

PubMed

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p < 0.01); a significant reduction of genome-wide DNA MLs ( p < 0.01); and an increase in the methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p < 0.01). Our study indicated that a reduction in folic acid concentration promotes DNA damage and DNA methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

Cumulative mtDNA damage and mutations contribute to the progressive loss of RGCs in a rat model of glaucoma

PubMed Central

Nickerson, John M.; Gao, Feng-juan; Sun, Zhongmou; Chen, Xin-ya; Zhang, Shu-jie; Gao, Feng; Chen, Jun-yi; Luo, Yi; Wang, Yan; Sun, Xing-huai

2015-01-01

Glaucoma is a chronic neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Mitochondrial DNA (mtDNA) alterations have been documented as a key component of many neurodegenerative disorders. However, whether mtDNA alterations contribute to the progressive loss of RGCs and the mechanism whereby this phenomenon could occur are poorly understood. We investigated mtDNA alterations in RGCs using a rat model of chronic intraocular hypertension and explored the mechanisms underlying progressive RGC loss. We demonstrate that the mtDNA damage and mutations triggered by intraocular pressure (IOP) elevation are initiating, crucial events in a cascade leading to progressive RGC loss. Damage to and mutation of mtDNA, mitochondrial dysfunction, reduced levels of mtDNA repair/replication enzymes, and elevated reactive oxygen species form a positive feedback loop that produces irreversible mtDNA damage and mutation and contributes to progressive RGC loss, which occurs even after a return to normal IOP. Furthermore, we demonstrate that mtDNA damage and mutations increase the vulnerability of RGCs to elevated IOP and glutamate levels, which are among the most common glaucoma insults. This study suggests that therapeutic approaches that target mtDNA maintenance and repair and that promote energy production may prevent the progressive death of RGCs. PMID:25478814

The human intra-S checkpoint response to UVC-induced DNA damage.

PubMed

Kaufmann, William K

2010-05-01

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC and ionizing radiation-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone photoproducts in DNA trigger the response is discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in Phaseolus vulgaris.

PubMed

Taspinar, Mahmut Sinan; Aydin, Murat; Sigmaz, Burcu; Yildirim, Nalan; Agar, Guleray

2017-10-01

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES

EPA Science Inventory

A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) ra...

DNA Damage Repair Genes Controlling Human Papillomavirus (HPV) Episome Levels under Conditions of Stability and Extreme Instability

PubMed Central

Edwards, Terri G.; Vidmar, Thomas J.; Koeller, Kevin; Bashkin, James K.; Fisher, Chris

2013-01-01

DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ∼5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process. PMID:24098381

The Cell's Sophisticated Army to Defend Against Assaults on DNAThe Cell's Sophisticated Army to Defend Against Assaults on DNA | Center for Cancer Research

Cancer.gov

The maintenance of genome integrity and function is essen-tial for the survival of cells and organisms. Any damage to our genetic material must be immediately sensed and repaired to preserve a cell’s func-tional integrity. Cells are constantly faced with the challenge of protecting their DNA from assaults by damaging chemicals and ultraviolet light. DNA damage that escapes repair can lead to a variety of genetic disorders and diseases, particularly cancer. To avoid this catastrophe, the cell employs an army of DNA repair factors that “rush to the scene” and initiate a cascade of events to repair the damage. Exactly how different repair factors sense DNA damage and orchestrate their concert-ed response is not well understood.

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription

PubMed Central

Aydin, Özge Z.; Marteijn, Jurgen A.; Ribeiro-Silva, Cristina; Rodríguez López, Aida; Wijgers, Nils; Smeenk, Godelieve; van Attikum, Haico; Poot, Raymond A.; Vermeulen, Wim; Lans, Hannes

2014-01-01

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA. PMID:24990377

ATM-Dependent Phosphorylation of MEF2D Promotes Neuronal Survival after DNA Damage

PubMed Central

Chan, Shing Fai; Sances, Sam; Brill, Laurence M.; Okamoto, Shu-ichi; Zaidi, Rameez; McKercher, Scott R.; Akhtar, Mohd W.; Nakanishi, Nobuki

2014-01-01

Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT. PMID:24672010

Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

PubMed Central

Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

2011-01-01

Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AX (γH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.

PubMed

Ollero, M; Gil-Guzman, E; Lopez, M C; Sharma, R K; Agarwal, A; Larson, K; Evenson, D; Thomas, A J; Alvarez, J G

2001-09-01

Reactive oxygen species (ROS)-induced damage of membrane phospholipids and DNA in human spermatozoa has been implicated in the pathogenesis of male infertility. In this study, variations in ROS production, DNA structure (as measured by the sperm chromatin structure assay) and lipid composition, were studied in human spermatozoa at different stages of maturation. Sperm subsets were isolated by discontinuous density gradient centrifugation of semen samples obtained from healthy donors and from infertility patients. DNA damage and ROS production were highest in immature spermatozoa with cytoplasmic retention and abnormal head morphology, and lowest in mature spermatozoa. Docosahexaenoic acid and sterol content were highest in immature germ cells and immature spermatozoa, and lowest in mature spermatozoa. The relative proportion of ROS-producing immature spermatozoa in the sample was directly correlated with DNA damage in mature spermatozoa, and inversely correlated with the recovery of motile spermatozoa. There was no correlation between DNA damage and sperm morphology in mature spermatozoa. The high levels of ROS production and DNA damage observed in immature spermatozoa may be indicative of derangements in the regulation of spermiogenesis. DNA damage in mature spermatozoa may be the result of oxidative damage by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis.

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

PubMed

Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

2015-07-01

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

Animal Studies in the Mode of Action of Agents, That Are Antitransformers in Cell Cultures.

DTIC Science & Technology

1987-10-28

The oel- let was hydrolysed at 90 C in 6% PCA for 30 min. The DNA content (ootical density at 260 nm and 290 nm) and the radioactivitv ( liquid ...required: DNA damage, excision of the damage and DNA-strand polimerization and ligation. The misrepair or incomplete repair of DNA damage may be an ini...with non ionic deter- gents in the ?resence of high salt concentration. The secondary and tertiary structure (supercoils) of DNA remains intact under

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

PubMed

Mo, Charlie Y; Culyba, Matthew J; Selwood, Trevor; Kubiak, Jeffrey M; Hostetler, Zachary M; Jurewicz, Anthony J; Keller, Paul M; Pope, Andrew J; Quinn, Amy; Schneck, Jessica; Widdowson, Katherine L; Kohli, Rahul M

2018-03-09

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.

Photo-induced Leishmania DNA degradation by silver-doped zinc oxide nanoparticle: an in-vitro approach.

PubMed

Nadhman, Akhtar; Sirajuddin, Muhammad; Nazir, Samina; Yasinzai, Masoom

2016-06-01

Recently, the authors reported newly synthesised polyethylene glycol (PEG)ylated silver (9%)-doped zinc oxide nanoparticle (doped semiconductor nanoparticle (DSN)) which has high potency for killing Leishmania tropica by producing reactive oxygen species on exposure to sunlight. The current report is focused on Leishmania DNA interaction and damage caused by the DSN. Here, we showed that the damage to Leishmania DNA was indirect, as the DSN was unable to interact with the DNA in intact Leishmania cell, indicating the incapability of PEGylated DSN to cross the nucleus barrier. The DNA damage was the result of high production of singlet oxygen on exposure to sunlight. The DNA damage was successfully prevented by singlet oxygen scavenger (sodium azide) confirming involvement of the highly energetic singlet oxygen in the DNA degradation process.

XRCC1 Arg399Gln was associated with repair capacity for DNA damage induced by occupational chromium exposure

PubMed Central

2012-01-01

Background Occupational chromium exposure may induce DNA damage and lead to lung cancer and other work-related diseases. DNA repair gene polymorphisms, which may alter the efficiency of DNA repair, thus may contribute to genetic susceptibility of DNA damage. The aim of this study was to test the hypothesis that the genetic variations of 9 major DNA repair genes could modulate the hexavalent chromium (Cr (VI))-induced DNA damage. Findings The median (P25-P75) of Olive tail moment was 0.93 (0.58–1.79) for individuals carrying GG genotype of XRCC1 Arg399Gln (G/A), 0.73 (0.46–1.35) for GA heterozygote and 0.50 (0.43–0.93) for AA genotype. Significant difference was found among the subjects with three different genotypes (P = 0.048) after adjusting the confounding factors. The median of Olive tail moment of the subjects carrying A allele (the genotypes of AA and GA) was 0.66 (0.44–1.31), which was significantly lower than that of subjects with GG genotype (P = 0.043). The A allele conferred a significantly reduced risk of DNA damage with the OR of 0.39 (95% CI: 0.15–0.99, P = 0.048). No significant association was found between the XRCC1Arg194Trp, ERCC1 C8092A, ERCC5 His1104Asp, ERCC6 Gly399Asp, GSTP1 Ile105Val, OGG1 Ser326Cys, XPC Lys939Gln, XPD Lys751Gln and DNA damage. Conclusion The polymorphism of Arg399Gln in XRCC1 was associated with the Cr (VI)- induced DNA damage. XRCC1 Arg399Gln may serve as a genetic biomarker of susceptibility for Cr (VI)- induced DNA damage. PMID:22642904

Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions*

PubMed Central

Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

2017-01-01

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se. Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. PMID:28302921

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions.

PubMed

Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

2017-05-01

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

AtPDCD5 Plays a Role in Programmed Cell Death after UV-B Exposure in Arabidopsis1[OPEN

PubMed Central

Falcone Ferreyra, María Lorena; D’Andrea, Lucio; AbdElgawad, Hamada

2016-01-01

DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs. PMID:26884483

Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

NASA Technical Reports Server (NTRS)

Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

2011-01-01

Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

PubMed Central

Lauretti, Elisabetta; Hulse, Michael; Siciliano, Micheal; Lupey-Green, Lena N.; Abraham, Aaron; Skorski, Tomasz; Tempera, Italo

2018-01-01

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers. PMID:29535829

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides.

PubMed

Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

2011-11-15

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p<0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37±2.15 vs. 6.24±1.37 tail% DNA, p<0.001). Further, the workers with CYP2D6*3PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p<0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. Copyright © 2011 Elsevier Inc. All rights reserved.

NF-κB inhibition delays DNA damage–induced senescence and aging in mice

PubMed Central

Tilstra, Jeremy S.; Robinson, Andria R.; Wang, Jin; Gregg, Siobhán Q.; Clauson, Cheryl L.; Reay, Daniel P.; Nasto, Luigi A.; St Croix, Claudette M.; Usas, Arvydas; Vo, Nam; Huard, Johnny; Clemens, Paula R.; Stolz, Donna B.; Guttridge, Denis C.; Watkins, Simon C.; Garinis, George A.; Wang, Yinsheng; Niedernhofer, Laura J.; Robbins, Paul D.

2012-01-01

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB–activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging. PMID:22706308

Magnesium Supplementation Diminishes Peripheral Blood Lymphocyte DNA Oxidative Damage in Athletes and Sedentary Young Man

PubMed Central

Petrović, Jelena; Stanić, Dušanka; Dmitrašinović, Gordana; Plećaš-Solarović, Bosiljka; Ignjatović, Svetlana; Batinić, Bojan; Popović, Dejana

2016-01-01

Sedentary lifestyle is highly associated with increased risk of cardiovascular disease, obesity, and type 2 diabetes. It is known that regular physical activity has positive effects on health; however several studies have shown that acute and strenuous exercise can induce oxidative stress and lead to DNA damage. As magnesium is essential in maintaining DNA integrity, the aim of this study was to determine whether four-week-long magnesium supplementation in students with sedentary lifestyle and rugby players could prevent or diminish impairment of DNA. By using the comet assay, our study demonstrated that the number of peripheral blood lymphocytes (PBL) with basal endogenous DNA damage is significantly higher in rugby players compared to students with sedentary lifestyle. On the other hand, magnesium supplementation significantly decreased the number of cells with high DNA damage, in the presence of exogenous H2O2, in PBL from both students and rugby players, and markedly reduced the number of cells with medium DNA damage in rugby players compared to corresponding control nonsupplemented group. Accordingly, the results of our study suggest that four-week-long magnesium supplementation has marked effects in protecting the DNA from oxidative damage in both rugby players and in young men with sedentary lifestyle. Clinical trial is registered at ANZCTR Trial Id: ACTRN12615001237572. PMID:27042258

Dynamic changes to survivin subcellular localization are initiated by DNA damage

PubMed Central

Asumen, Maritess Gay; Ifeacho, Tochukwu V; Cockerham, Luke; Pfandl, Christina; Wall, Nathan R

2010-01-01

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality. PMID:20856848