Glutathione-deficient Plasmodium berghei parasites exhibit growth delay and nuclear DNA damage.

PubMed

Padín-Irizarry, Vivian; Colón-Lorenzo, Emilee E; Vega-Rodríguez, Joel; Castro, María Del R; González-Méndez, Ricardo; Ayala-Peña, Sylvette; Serrano, Adelfa E

2016-06-01

Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

Checkpoints: It takes more than time to heal some wounds

PubMed Central

Rhind, Nicholas; Russell, Paul

2010-01-01

The S-phase DNA damage checkpoint seems to provide a twist on the checkpoint theme. Instead of delaying replication and allowing repair as a consequence, it may activate repair and delay replication as a consequence. PMID:11137027

DNA double strand break repair defect and sensitivity to poly ADP-ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma

PubMed Central

Weaver, Alice N.; Cooper, Tiffiny S.; Rodriguez, Marcela; Trummell, Hoa Q.; Bonner, James A.; Rosenthal, Eben L.; Yang, Eddy S.

2015-01-01

Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV− HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10–14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC. PMID:26336991

Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

PubMed

Wolf, Norman; Pendergrass, William; Singh, Narendra; Swisshelm, Karen; Schwartz, Jeffrey

2008-02-05

This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts. Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses. This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes. The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress.

PubMed

Amaral, Nuno; Vendrell, Alexandre; Funaya, Charlotta; Idrissi, Fatima-Zahra; Maier, Michael; Kumar, Arun; Neurohr, Gabriel; Colomina, Neus; Torres-Rosell, Jordi; Geli, María-Isabel; Mendoza, Manuel

2016-05-01

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.

Recovery from DNA damage-induced G2 arrest requires actin-binding protein filamin-A/actin-binding protein 280.

PubMed

Meng, Xiangbing; Yuan, Yuan; Maestas, Adrian; Shen, Zhiyuan

2004-02-13

Filamin-A (filamin-1) is an actin-binding protein involved in the organization of actin networks. Our previous study shows that filamin-A interacts with BRCA2, and lack of filamin-A expression results in increased cellular sensitivity to several DNA damaging agents in melanoma cells (Yuan, Y., and Shen, Z. (2001) J. Biol. Chem. 276, 48318-48324), suggesting a role of filamin-A in DNA damage response. In this report, we demonstrated that deficiency of filamin-A results in an 8-h delay in the recovery from G2 arrest in response to ionizing radiation. However, filamin-A deficiency does not affect the initial activation of the G2/M checkpoint. We also found that filamin-A deficiency results in sustained activation of Chk1 and Chk2 after irradiation. This in turn causes a delay in the dephosphorylation of phospho-Cdc2, which is inhibitory to the G2/M transition. In addition, filamin-A-deficient M2 cells undergo mitotic catastrophe-related nuclear fragmentation after they are released from the G2 arrest. Together, these data suggest a functional role of filamin-A in the recovery from G2 arrest and subsequent mitotic cell death after DNA damage.

Nucleotide excision repair deficient mouse models and neurological disease

PubMed Central

Niedernhofer, Laura J.

2008-01-01

Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA base damage. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human diseases caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated aging. All three syndromes include neurological disease, indicating an important role for NER in protecting against spontaneous DNA damage as well. To study the pathophysiology caused by DNA damage, numerous mouse models of NER deficiency were generated by knocking-out genes required for NER or knocking-in disease-causing human mutations. This review explores the utility of these mouse models to study neurological disease caused by NER deficiency. PMID:18272436

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

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

Acute Doxorubicin Insult in the Mouse Ovary Is Cell- and Follicle-Type Dependent

PubMed Central

Roti Roti, Elon C.; Leisman, Scott K.; Abbott, David H.; Salih, Sana M.

2012-01-01

Primary ovarian insufficiency (POI) is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR) as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours) than granulosa cells (measurable at 4 hours), as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10–12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult in the ovary must act almost immediately to prevent acute insult as significant damage was seen in stroma cells within the first two hours. PMID:22876313

XRN2 Links Transcription Termination to DNA Damage and Replication Stress

PubMed Central

Patidar, Praveen L.; Motea, Edward A.; Dang, Tuyen T.; Manley, James L.

2016-01-01

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability. PMID:27437695

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

PubMed

Morales, Julio C; Richard, Patricia; Patidar, Praveen L; Motea, Edward A; Dang, Tuyen T; Manley, James L; Boothman, David A

2016-07-01

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

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

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

Protein phosphatase 2A inhibition enhances radiation sensitivity and reduces tumor growth in chordoma.

PubMed

Hao, Shuyu; Song, Hua; Zhang, Wei; Seldomridge, Ashlee; Jung, Jinkyu; Giles, Amber J; Hutchinson, Marsha-Kay; Cao, Xiaoyu; Colwell, Nicole; Lita, Adrian; Larion, Mioara; Maric, Dragan; Abu-Asab, Mones; Quezado, Martha; Kramp, Tamalee; Camphausen, Kevin; Zhuang, Zhengping; Gilbert, Mark R; Park, Deric M

2018-05-18

Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

PubMed

Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

2015-03-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

PubMed Central

Colin, Didier J.; Hain, Karolina O.; Allan, Lindsey A.; Clarke, Paul R.

2015-01-01

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies. PMID:25761368

Activation of a yeast replication origin near a double-stranded DNA break.

PubMed

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

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).

β2-spectrin depletion impairs DNA damage repair

PubMed Central

Horikoshi, Nobuo; Pandita, Raj K.; Mujoo, Kalpana; Hambarde, Shashank; Sharma, Dharmendra; Mattoo, Abid R.; Chakraborty, Sharmistha; Charaka, Vijaya; Hunt, Clayton R.; Pandita, Tej K.

2016-01-01

β2-Spectrin (β2SP/SPTBN1, gene SPTBN1) is a key TGF-β/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-β signaling and can contribute to liver cancer development. Here we report that cells deficient in β2-Spectrin (β2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), β2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, β2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in β2SP deficient cells. We propose that β2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination. PMID:27248179

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

PubMed Central

Mund, Andreas; Schubert, Tobias; Staege, Hannah; Kinkley, Sarah; Reumann, Kerstin; Kriegs, Malte; Fritsch, Lauriane; Battisti, Valentine; Ait-Si-Ali, Slimane; Hoffbeck, Anne-Sophie; Soutoglou, Evi; Will, Hans

2012-01-01

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure. PMID:23034801

Mitochondrial Enzyme Plays Critical Role in Chemotherapy-Induced Heart Damage | Center for Cancer Research

Cancer.gov

Doxorubicin (DOX) is an effective drug for treating cancers ranging from leukemia and lymphoma to solid tumors, such as breast cancer. DOX kills dividing cells in two ways: inserting between the base pairs of DNA and trapping a complex of DNA and an enzyme that cuts DNA, topoisomerase 2α, preventing DNA repair. However, DOX also causes congestive heart failure in about 30 percent of adult cancer patients and delayed onset heart failure in a significant number of pediatric cancer patients. The mechanism of this DOX-mediated cardiotoxicity is not well understood since heart muscle cells neither divide nor express Top2α, and there are currently no genetic factors that identify patients who are susceptible to cardiac damage from DOX. However, a recent study showed that mice lacking another topoisomerase, Top2β, did not experience cardiac damage after treatment with DOX.

ATM-dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

PubMed Central

Zhou, Tong; Chou, Jeff; Zhou, Yingchun; Simpson, Dennis A.; Cao, Feng; Bushel, Pierre R.; Paules, Richard S.; Kaufmann, William K.

2013-01-01

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. PMID:17699107

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.

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

PubMed

Ahmed, Emad A; Scherthan, Harry; de Rooij, Dirk G

2015-12-16

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores.

PubMed

Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z; Zhong, Hui; Cao, Cheng; Xu, Quanbin

2013-04-15

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.

Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

PubMed Central

Arango, Daniel; Parihar, Arti; Villamena, Frederick A.; Wang, Liwen; Freitas, Michael A.; Grotewold, Erich; Doseff, Andrea I.

2014-01-01

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid. PMID:22985621

DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds

PubMed Central

Waterworth, Wanda M.; Footitt, Steven; Bray, Clifford M.; Finch-Savage, William E.; West, Christopher E.

2016-01-01

Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production. PMID:27503884

DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

PubMed

Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

2016-08-23

Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

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

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

PubMed Central

Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

2016-01-01

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

Short and long time effects of low temperature Plasma Activated Media on 3D multicellular tumor spheroids

NASA Astrophysics Data System (ADS)

Judée, Florian; Fongia, Céline; Ducommun, Bernard; Yousfi, Mohammed; Lobjois, Valérie; Merbahi, Nofel

2016-02-01

This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H2O2 plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that and OH* are involved in H2O2 formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or -80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors.

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

PubMed

Siede, W; Friedberg, A S; Friedberg, E C

1993-09-01

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

Anthocyanins Delay Ageing-Related Degenerative Changes in the Liver.

PubMed

Wei, Jie; Zhang, Guokun; Zhang, Xiao; Xu, Dexin; Gao, Jun; Fan, Jungang

2017-12-01

Liver ageing is a significant risk factor for chronic liver diseases. Anthocyanin is a food additive that has previously shown efficacy in increasing longevity. Here, we tested whether anthocyanins could protect young mice from accelerated ageing of the liver. Kunming mice were injected with D-galactose to accelerate ageing and were given 20 or 40 mg/kg anthocyanins as an intervention. After eight weeks, whole liver function and structure were evaluated, and the expression levels of genes involved in the DNA damage signalling pathway were assessed by Western blot analysis. Anthocyanins delayed the reduction of the liver index (p < 0.05), hepatic tissue injury and fibrosis. Anthocyanins also maintained the stability of the redox system (GSH-PX, T-SOD and MDA) in plasma and liver structures (p < 0.001) and reduced the levels of inflammatory factors (IL-1, IL-6 and TNF-α) in the liver (p < 0.05). Moreover, the expression levels of sensors (ATM and ATR), mediators (H2AX and γ-H2AX) and effectors (Chk1, Chk2, p53 and p-p53) in the DNA damage signalling pathway were all reduced. Anthocyanins could be widely used in the field of health products to slow ageing-related deterioration of liver function and structure by inhibiting DNA damage.

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

PubMed Central

Kim, Seung-Hwan; Holway, Antonia H.; Wolff, Suzanne; Dillin, Andrew; Michael, W. Matthew

2007-01-01

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context. PMID:17908915

Urea transporter UT-B deletion induces DNA damage and apoptosis in mouse bladder urothelium.

PubMed

Dong, Zixun; Ran, Jianhua; Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders.

Urea Transporter UT-B Deletion Induces DNA Damage and Apoptosis in Mouse Bladder Urothelium

PubMed Central

Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Background Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Methodology/Principal Findings Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. Conclusions/Significance UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders. PMID:24204711

Effects of early life exposure to ultraviolet C radiation on mitochondrial DNA content, transcription, ATP production, and oxygen consumption in developing Caenorhabditis elegans

PubMed Central

2013-01-01

Background Mitochondrial DNA (mtDNA) is present in multiple copies per cell and undergoes dramatic amplification during development. The impacts of mtDNA damage incurred early in development are not well understood, especially in the case of types of mtDNA damage that are irreparable, such as ultraviolet C radiation (UVC)-induced photodimers. Methods We exposed first larval stage nematodes to UVC using a protocol that results in accumulated mtDNA damage but permits nuclear DNA (nDNA) repair. We then measured the transcriptional response, as well as oxygen consumption, ATP levels, and mtDNA copy number through adulthood. Results Although the mtDNA damage persisted to the fourth larval stage, we observed only a relatively minor ~40% decrease in mtDNA copy number. Transcriptomic analysis suggested an inhibition of aerobic metabolism and developmental processes; mRNA levels for mtDNA-encoded genes were reduced ~50% at 3 hours post-treatment, but recovered and, in some cases, were upregulated at 24 and 48 hours post-exposure. The mtDNA polymerase γ was also induced ~8-fold at 48 hours post-exposure. Moreover, ATP levels and oxygen consumption were reduced in response to UVC exposure, with marked reductions of ~50% at the later larval stages. Conclusions These results support the hypothesis that early life exposure to mitochondrial genotoxicants could result in mitochondrial dysfunction at later stages of life, thereby highlighting the potential health hazards of time-delayed effects of these genotoxicants in the environment. PMID:23374645

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores

PubMed Central

Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z.; Zhong, Hui; Cao, Cheng; Xu, Quanbin

2013-01-01

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase. PMID:23531678

DNA Damage as a Driver for Growth Delay: Chromosome Instability Syndromes with Intrauterine Growth Retardation

PubMed Central

Hernández-Gómez, Mariana

2017-01-01

DNA is constantly exposed to endogenous and exogenous mutagenic stimuli that are capable of producing diverse lesions. In order to protect the integrity of the genetic material, a wide array of DNA repair systems that can target each specific lesion has evolved. Despite the availability of several repair pathways, a common general program known as the DNA damage response (DDR) is stimulated to promote lesion detection, signaling, and repair in order to maintain genetic integrity. The genes that participate in these pathways are subject to mutation; a loss in their function would result in impaired DNA repair and genomic instability. When the DDR is constitutionally altered, every cell of the organism, starting from development, will show DNA damage and subsequent genomic instability. The cellular response to this is either uncontrolled proliferation and cell cycle deregulation that ensues overgrowth, or apoptosis and senescence that result in tissue hypoplasia. These diverging growth abnormalities can clinically translate as cancer or growth retardation; both features can be found in chromosome instability syndromes (CIS). The analysis of the clinical, cellular, and molecular phenotypes of CIS with intrauterine growth retardation allows inferring that replication alteration is their unifying feature. PMID:29238724

Radiation-Induced Epigenetic Alterations after Low and High LET Irradiations

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

Aypar, Umut; Morgan, William F.; Baulch, Janet E.

Epigenetics, including DNA methylation and microRNA (miRNA) expression, could be the missing link in understanding the delayed, non-targeted effects of radiation including radiationinduced genomic instability (RIGI). This study tests the hypothesis that irradiation induces epigenetic aberrations, which could eventually lead to RIGI, and that the epigenetic aberrations induced by low linear energy transfer (LET) irradiation are different than those induced by high LET irradiations. GM10115 cells were irradiated with low LET x-rays and high LET iron (Fe) ions and evaluated for DNA damage, cell survival and chromosomal instability. The cells were also evaluated for specific locus methylation of nuclear factor-kappamore » B (NFκB), tumor suppressor in lung cancer 1 (TSLC1) and cadherin 1 (CDH1) gene promoter regions, long interspersed nuclear element 1 (LINE-1) and Alu repeat element methylation, CpG and non-CpG global methylation and miRNA expression levels. Irradiated cells showed increased micronucleus induction and cell killing immediately following exposure, but were chromosomally stable at delayed times post-irradiation. At this same delayed time, alterations in repeat element and global DNA methylation and miRNA expression were observed. Analyses of DNA methylation predominantly showed hypomethylation, however hypermethylation was also observed. MiRNA shown to be altered in expression level after x-ray irradiation are involved in chromatin remodeling and DNA methylation. Different and higher incidence of epigenetic changes were observed after exposure to low LET x-rays than high LET Fe ions even though Fe ions elicited more chromosomal damage and cell killing. This study also shows that the irradiated cells acquire epigenetic changes even though they are chromosomally stable suggesting that epigenetic aberrations may arise in the cell without initiating RIGI.« less

Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

PubMed

Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

2018-03-05

Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response.

PubMed

Salton, Maayan; Lerenthal, Yaniv; Wang, Shih-Ya; Chen, David J; Shiloh, Yosef

2010-04-15

The DNA damage response (DDR) is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which phosphorylates key players in its various branches. SFPQ (PSF) and NONO (p54) are nuclear proteins that interact with each other and have diverse roles in nucleic acids metabolism. The SFPQ/NONO heterodimer was previously found to enhance DNA strand break rejoining in vitro. Our attention was drawn to these two proteins as they interact with the nuclear matrix protein Matrin 3 (MATR3), which we found to be a novel ATM target. We asked whether SFPQ and NONO too are involved in the DSB response. Proteins that function at the early phase of this response are often recruited to the damaged sites. We observed rapid recruitment of SFPQ/NONO to sites of DNA damage induced by laser microbeam. In MATR3 knockdown cells SFPQ/NONO retention at DNA damage sites was prolonged. SFPQ and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle following treatment with the radiomimetic chemical neocarzinostatin. Notably, proteins involved in DSB repair via nonhomologous end-joining co-immunoprecipitated with NONO; SFPQ depletion delayed DSB repair. Collectively the data suggest that SFPQ, NONO and MATR3 are involved in the early stage of the DSB response, setting the scene for DSB repair.

Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

PubMed Central

2013-01-01

Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway. PMID:23347679

Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

2013-01-24

In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.

Cytoplasmic localization of Hug1p, a negative regulator of the MEC1 pathway, coincides with the compartmentalization of Rnr2p–Rnr4p

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

Ainsworth, William B.; Hughes, Bridget Todd; Au, Wei Chun

2013-10-04

Highlights: •Hug1p overexpression sensitizes wild-type cells to DNA damage and hydroxyurea (HU). •Expression of Hug1p in response to HU treatment is delayed relative to Rnr3p. •MEC1 pathway genes are required for cytoplasmic localization of Hug1p. •Hug1p subcellular compartmentalization to the cytoplasm coincides with Rnr2p–Rnr4p. -- Abstract: The evolutionarily conserved MEC1 checkpoint pathway mediates cell cycle arrest and induction of genes including the RNR (Ribonucleotide reductase) genes and HUG1 (Hydroxyurea, ultraviolet, and gamma radiation) in response to DNA damage and replication arrest. Rnr complex activity is in part controlled by cytoplasmic localization of the Rnr2p–Rnr4p subunits and inactivation of negative regulatorsmore » Sml1p and Dif1p upon DNA damage and hydroxyurea (HU) treatment. We previously showed that a deletion of HUG1 rescues lethality of mec1Δ and suppresses dun1Δ strains. In this study, multiple approaches demonstrate the regulatory response of Hug1p to DNA damage and HU treatment and support its role as a negative effector of the MEC1 pathway. Consistent with our hypothesis, wild-type cells are sensitive to DNA damage and HU when HUG1 is overexpressed. A Hug1 polyclonal antiserum reveals that HUG1 encodes a protein in budding yeast and its MEC1-dependent expression is delayed compared to the rapid induction of Rnr3p in response to HU treatment. Cell biology and subcellular fractionation experiments show localization of Hug1p-GFP to the cytoplasm upon HU treatment. The cytoplasmic localization of Hug1p-GFP is dependent on MEC1 pathway genes and coincides with the cytoplasmic localization of Rnr2p–Rnr4p. Taken together, the genetic interactions, gene expression, and localization studies support a novel role for Hug1p as a negative regulator of the MEC1 checkpoint response through its compartmentalization with Rnr2p–Rnr4p.« less

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

PubMed Central

Singh, Mayank; Hunt, Clayton R.; Pandita, Raj K.; Kumar, Rakesh; Yang, Chin-Rang; Horikoshi, Nobuo; Bachoo, Robert; Serag, Sara; Story, Michael D.; Shay, Jerry W.; Powell, Simon N.; Gupta, Arun; Jeffery, Jessie; Pandita, Shruti; Chen, Benjamin P. C.; Deckbar, Dorothee; Löbrich, Markus; Yang, Qin; Khanna, Kum Kum; Worman, Howard J.

2013-01-01

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair. PMID:23319047

N-Acetyl-L-cysteine protects thyroid cells against DNA damage induced by external and internal irradiation.

PubMed

Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

2017-11-01

We evaluated the effect of the antioxidant N-acetyl-L-cysteine (NAC) on the levels of reactive oxygen species (ROS), DNA double strand breaks (DSB) and micronuclei (MN) induced by internal and external irradiation using a rat thyroid cell line PCCL3. In internal irradiation experiments, ROS and DSB levels increased immediately after 131 I addition and then gradually declined, resulting in very high levels of MN at 24 and 48 h. NAC administration both pre- and also post- 131 I addition suppressed ROS, DSB and MN. In external irradiation experiments with a low dose (0.5 Gy), ROS and DSB increased shortly and could be prevented by NAC administration pre-, but not post-irradiation. In contrast, external irradiation with a high dose (5 Gy) increased ROS and DSB in a bimodal way: ROS and DSB levels increased immediately after irradiation, quickly returned to the basal levels and gradually rose again after >24 h. The second phase was in parallel with an increase in 4-hydroxy-2-nonenal. The number of MN induced by the second wave of ROS/DSB elevations was much higher than that by the first peak. In this situation, NAC administered pre- and post-irradiation comparably suppressed MN induced by a delayed ROS elevation. In conclusion, a prolonged ROS increase during internal irradiation and a delayed ROS increase after external irradiation with a high dose caused serious DNA damage, which were efficiently prevented by NAC. Thus, NAC administration even both after internal or external irradiation prevents ROS increase and eventual DNA damage.

Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1.

PubMed

Boddy, M N; Lopez-Girona, A; Shanahan, P; Interthal, H; Heyer, W D; Russell, P

2000-12-01

Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.

Impaired tRNA nuclear export links DNA damage and cell-cycle checkpoint.

PubMed

Ghavidel, Ata; Kislinger, Thomas; Pogoutse, Oxana; Sopko, Richelle; Jurisica, Igor; Emili, Andrew

2007-11-30

In response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression.

Tumour-cell apoptosis after cisplatin treatment is not telomere dependent.

PubMed

Jeyapalan, Jessie C; Saretzki, Gabriele; Leake, Alan; Tilby, Michael J; von Zglinicki, Thomas

2006-06-01

Cisplatin is a major chemotherapeutic agent, especially for the treatment of neuroblastoma. Telomeres with their sequence (TTAGGG)n are probable targets for cisplatin intrastrand cross-linking, but the role of telomeres in mediating cisplatin cytotoxicity is not clear. After exposure to cisplatin as single dose or continuous treatment, we found no loss of telomeres in either SHSY5Y neuroblastoma cells (telomere length, approximately 4 kbp), HeLa 229 cells (telomere length, 20 kbp) or in the acute lymphoblastic T cell line 1301 (telomere length, approximately 80 kbp). There was no induction of telomeric single strand breaks, telomeric overhangs were not degraded and telomerase activity was down-regulated only after massive onset of apoptosis. In contrast, cisplatin induced a delayed formation of DNA strand breaks and induced DNA damage foci containing gamma-H2A.X at nontelomeric sites. Interstitial DNA damage appears to be more important than telomere loss or telomeric damage as inducer of the signal pathway towards apoptosis and/or growth arrest in cisplatin-treated tumour cells.

Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage.

PubMed

Bayart, Emilie; Pouzoulet, Frédéric; Calmels, Lucie; Dadoun, Jonathan; Allot, Fabien; Plagnard, Johann; Ravanat, Jean-Luc; Bridier, André; Denozière, Marc; Bourhis, Jean; Deutsch, Eric

2017-01-01

Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2'-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 μM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy.

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

PubMed Central

Moseley, B. E. B.

1969-01-01

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision. PMID:5773016

Radiation induced genome instability: multiscale modelling and data analysis

NASA Astrophysics Data System (ADS)

Andreev, Sergey; Eidelman, Yuri

2012-07-01

Genome instability (GI) is thought to be an important step in cancer induction and progression. Radiation induced GI is usually defined as genome alterations in the progeny of irradiated cells. The aim of this report is to demonstrate an opportunity for integrative analysis of radiation induced GI on the basis of multiscale modelling. Integrative, systems level modelling is necessary to assess different pathways resulting in GI in which a variety of genetic and epigenetic processes are involved. The multilevel modelling includes the Monte Carlo based simulation of several key processes involved in GI: DNA double strand breaks (DSBs) generation in cells initially irradiated as well as in descendants of irradiated cells, damage transmission through mitosis. Taking the cell-cycle-dependent generation of DNA/chromosome breakage into account ensures an advantage in estimating the contribution of different DNA damage response pathways to GI, as to nonhomologous vs homologous recombination repair mechanisms, the role of DSBs at telomeres or interstitial chromosomal sites, etc. The preliminary estimates show that both telomeric and non-telomeric DSB interactions are involved in delayed effects of radiation although differentially for different cell types. The computational experiments provide the data on the wide spectrum of GI endpoints (dicentrics, micronuclei, nonclonal translocations, chromatid exchanges, chromosome fragments) similar to those obtained experimentally for various cell lines under various experimental conditions. The modelling based analysis of experimental data demonstrates that radiation induced GI may be viewed as processes of delayed DSB induction/interaction/transmission being a key for quantification of GI. On the other hand, this conclusion is not sufficient to understand GI as a whole because factors of DNA non-damaging origin can also induce GI. Additionally, new data on induced pluripotent stem cells reveal that GI is acquired in normal mature cells during genome reprogramming by the oncogene c-myc and three additional transcription factors. These and other data reveal the need for generalisation of current model of GI. One can expect that different early events of both DNA damaging and non-damaging origins merge in a single late pathway. To search for a deeper view we propose to redefine GI as genome destabilisation manifested in erosion of genome states and altered transitions between states. This changing view on GI may help to integrate the inducing factors of various origins in the single basic model of GI.

NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

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

Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi

2015-06-05

Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated inmore » the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.« less

Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells.

PubMed

Lefevre, Sophie; Brossas, Caroline; Auchère, Françoise; Boggetto, Nicole; Camadro, Jean-Michel; Santos, Renata

2012-09-15

Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H(2)O(2). On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.

Transcription Factor RFX1 Is Crucial for Maintenance of Genome Integrity in Fusarium graminearum

PubMed Central

Min, Kyunghun; Son, Hokyoung; Lim, Jae Yun; Choi, Gyung Ja; Kim, Jin-Cheol; Harris, Steven D.

2014-01-01

The survival of cellular organisms depends on the faithful replication and transmission of DNA. Regulatory factor X (RFX) transcription factors are well conserved in animals and fungi, but their functions are diverse, ranging from the DNA damage response to ciliary gene regulation. We investigated the role of the sole RFX transcription factor, RFX1, in the plant-pathogenic fungus Fusarium graminearum. Deletion of rfx1 resulted in multiple defects in hyphal growth, conidiation, virulence, and sexual development. Deletion mutants of rfx1 were more sensitive to various types of DNA damage than the wild-type strain. Septum formation was inhibited and micronuclei were produced in the rfx1 deletion mutants. The results of the neutral comet assay demonstrated that disruption of rfx1 function caused spontaneous DNA double-strand breaks (DSBs). The transcript levels of genes involved in DNA DSB repair were upregulated in the rfx1 deletion mutants. DNA DSBs produced micronuclei and delayed septum formation in F. graminearum. Green fluorescent protein (GFP)-tagged RFX1 localized in nuclei and exhibited high expression levels in growing hyphae and conidiophores, where nuclear division was actively occurring. RNA-sequencing-based transcriptomic analysis revealed that RFX1 suppressed the expression of many genes, including those required for the repair of DNA damage. Taken together, these findings indicate that the transcriptional repressor rfx1 performs crucial roles during normal cell growth by maintaining genome integrity. PMID:24465002

Metabolic interrogation as a tool to optimize chemotherapeutic regimens.

PubMed

Sandulache, Vlad C; Chen, Yunyun; Feng, Lei; William, William N; Skinner, Heath D; Myers, Jeffrey N; Meyn, Raymond E; Li, Jinzhong; Mijiti, Ainiwaer; Bankson, James A; Fuller, Clifton D; Konopleva, Marina Y; Lai, Stephen Y

2017-03-14

Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC).We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines.Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies.

Metabolic interrogation as a tool to optimize chemotherapeutic regimens

PubMed Central

Feng, Lei; William, William N.; Skinner, Heath D.; Myers, Jeffrey N.; Meyn, Raymond E.; Li, Jinzhong; Mijiti, Ainiwaer; Bankson, James A.; Fuller, Clifton D.; Konopleva, Marina Y.; Lai, Stephen Y.

2017-01-01

Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC). We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines. Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies. PMID:28184025

HAC1 and HAF1 Histone Acetyltransferases Have Different Roles in UV-B Responses in Arabidopsis.

PubMed

Fina, Julieta P; Masotti, Fiorella; Rius, Sebastián P; Crevacuore, Franco; Casati, Paula

2017-01-01

Arabidopsis has 12 histone acetyltransferases grouped in four families: the GNAT/HAG, the MYST/HAM, the p300/CBP/HAC and the TAFII250/HAF families. We previously showed that ham1 and ham2 mutants accumulated higher damaged DNA after UV-B exposure than WT plants. In contrast, hag3 RNA interference transgenic plants showed less DNA damage and lower inhibition of plant growth by UV-B, and increased levels of UV-B-absorbing compounds. These results demonstrated that HAM1, HAM2, and HAG3 participate in UV-B-induced DNA damage repair and signaling. In this work, to further explore the role of histone acetylation in UV-B responses, a putative function of other acetyltransferases of the HAC and the HAF families was analyzed. Neither HAC nor HAF acetyltrasferases participate in DNA damage and repair after UV-B radiation in Arabidopsis. Despite this, haf1 mutants presented lower inhibition of leaf and root growth by UV-B, with altered expression of E2F transcription factors. On the other hand, hac1 plants showed a delay in flowering time after UV-B exposure and changes in FLC and SOC1 expression patterns. Our data indicate that HAC1 and HAF1 have crucial roles for in UV-B signaling, confirming that, directly or indirectly, both enzymes also have a role in UV-B responses.

HAC1 and HAF1 Histone Acetyltransferases Have Different Roles in UV-B Responses in Arabidopsis

PubMed Central

Fina, Julieta P.; Masotti, Fiorella; Rius, Sebastián P.; Crevacuore, Franco; Casati, Paula

2017-01-01

Arabidopsis has 12 histone acetyltransferases grouped in four families: the GNAT/HAG, the MYST/HAM, the p300/CBP/HAC and the TAFII250/HAF families. We previously showed that ham1 and ham2 mutants accumulated higher damaged DNA after UV-B exposure than WT plants. In contrast, hag3 RNA interference transgenic plants showed less DNA damage and lower inhibition of plant growth by UV-B, and increased levels of UV-B-absorbing compounds. These results demonstrated that HAM1, HAM2, and HAG3 participate in UV-B-induced DNA damage repair and signaling. In this work, to further explore the role of histone acetylation in UV-B responses, a putative function of other acetyltransferases of the HAC and the HAF families was analyzed. Neither HAC nor HAF acetyltrasferases participate in DNA damage and repair after UV-B radiation in Arabidopsis. Despite this, haf1 mutants presented lower inhibition of leaf and root growth by UV-B, with altered expression of E2F transcription factors. On the other hand, hac1 plants showed a delay in flowering time after UV-B exposure and changes in FLC and SOC1 expression patterns. Our data indicate that HAC1 and HAF1 have crucial roles for in UV-B signaling, confirming that, directly or indirectly, both enzymes also have a role in UV-B responses. PMID:28740501

Fanconi anemia proteins in telomere maintenance.

PubMed

Sarkar, Jaya; Liu, Yie

2016-07-01

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell. Published by Elsevier B.V.

Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice.

PubMed

Zhang, Qing-Shuo; Tang, Weiliang; Deater, Matthew; Phan, Ngoc; Marcogliese, Andrea N; Li, Hui; Al-Dhalimy, Muhsen; Major, Angela; Olson, Susan; Monnat, Raymond J; Grompe, Markus

2016-12-15

Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2 -/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2 -/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2 -/- Trp53 +/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells. © 2016 by The American Society of Hematology.

Biophysical modelling of early and delayed radiation damage at chromosome level

NASA Astrophysics Data System (ADS)

Andreev, S.; Eidelman, Y.

Exposure by ionising radiation increases cancer risk in human population Cancer is thought to originate from an altered expression of certain number of specific genes It is now widely recognised that chromosome aberrations CA are involved in stable change in expression of genes by gain or loss of their functions Thus CA can contribute to initiation or progression of cancer Therefore understanding mechanisms of CA formation in the course of cancer development might be valuable tool for quantification and prognosis of different stages of radiation carcinogenesis Early CA are defined as aberrations induced in first post-irradiation mitotic cycle The present work describes the original biophysical technique for early CA modelling It includes the following simulation steps the ionising particle track structure the structural organisation of all chromosomes in G 0 G 1 cell nucleus spatial distribution of radiation induced DNA double-strand breaks dsb within chromosomes dsb rejoining and misrejoining modelling cell cycle taking into account mitotic delay which results in complex time dependence of aberrant cells in first mitosis The results on prediction of dose-response curves for simple and complex CA measured in cells undergoing first division cycle are presented in comparison with recent experimental data There is increasing evidence that CA are also observed in descendents of irradiated cells many generations after direct DNA damage These delayed CA or chromosome instability CI are thought to be a manifestation of genome

Sulfur mustard analog, 2-chloroethyl ethyl sulfide-induced skin injury involves DNA damage and induction of inflammatory mediators, in part via oxidative stress, in SKH-1 hairless mouse skin

PubMed Central

Jain, Anil K.; Tewari-Singh, Neera; Gu, Mallikarjuna; Inturi, Swetha; White, Carl W.; Agarwal, Rajesh

2011-01-01

Bifunctional alkyalating agent, Sulfur mustard (SM)-caused cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 or 4 mg CEES for 9–48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in COX-2, iNOS, and MMP-9 levels, indicating the involvement of DNA damage and inflammation in CEES-caused skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-caused DNA damage and the induction of inflammatory molecules. Oral GSH (300mg/kg) administration 1 h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injuries involve DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injuries in humans by SM. PMID:21722719

Indicators of replicative damage in equine tendon fibroblast monolayers

PubMed Central

2013-01-01

Background Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. Results We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents. Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress. Conclusions Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled. PMID:24025445

“Stockpile” of Slight Transcriptomic Changes Determines the Indirect Genotoxicity of Low-Dose BPA in Thyroid Cells

PubMed Central

Porreca, Immacolata; Ulloa Severino, Luisa; D’Angelo, Fulvio; Cuomo, Danila; Ceccarelli, Michele; Altucci, Lucia; Amendola, Elena; Nebbioso, Angela; Mallardo, Massimo

2016-01-01

Epidemiological and experimental data highlighted the thyroid-disrupting activity of bisphenol A (BPA). Although pivotal to identify the mechanisms of toxicity, direct low-dose BPA effects on thyrocytes have not been assessed. Here, we report the results of microarray experiments revealing that the transcriptome reacts dynamically to low-dose BPA exposure, adapting the changes in gene expression to the exposure duration. The response involves many genes, enriching specific pathways and biological functions mainly cell death/proliferation or DNA repair. Their expression is only slightly altered but, since they enrich specific pathways, this results in major effects as shown here for transcripts involved in the DNA repair pathway. Indeed, even though no phenotypic changes are induced by the treatment, we show that the exposure to BPA impairs the cell response to further stressors. We experimentally verify that prolonged exposure to low doses of BPA results in a delayed response to UV-C-induced DNA damage, due to impairment of p21-Tp53 axis, with the BPA-treated cells more prone to cell death and DNA damage accumulation. The present findings shed light on a possible mechanism by which BPA, not able to directly cause genetic damage at environmental dose, may exert an indirect genotoxic activity. PMID:26982218

Genotoxicity and Cytotoxicity Evaluation of the Neolignan Analogue 2-(4-Nitrophenoxy)-1Phenylethanone and its Protective Effect Against DNA Damage

PubMed Central

Hanusch, Alex Lucas; de Oliveira, Guilherme Roberto; de Sabóia-Morais, Simone Maria Teixeira; Machado, Rafael Cosme; dos Anjos, Murilo Machado; Chen Chen, Lee

2015-01-01

Neolignans are secondary metabolites found in various groups of Angiosperms. They belong to a class of natural compounds with great diversity of chemical structures and pharmacological activities. These compounds are formed by linking two phenylpropanoid units. Several compounds that have ability to prevent genetic damage have been isolated from plants, and can be used to prevent or delay the development of tumor cells. Genetic toxicology evaluation is widely used in risk assessment of new drugs in preclinical screening tests. In this study, we evaluated the genotoxicity and cytotoxicity of the neolignan analogue 2-(4-nitrophenoxy)-1-phenylethanone (4NF) and its protective effect against DNA damage using the mouse bone marrow micronucleus test and the comet assay in mouse peripheral blood. Our results showed that this neolignan analogue had no genotoxic activity and was able to reduce induced damage both in mouse bone marrow and peripheral blood. Although the neolignan analogue 4NF was cytotoxic, it reduced cyclophosphamide-induced cytotoxicity. In conclusion, it showed no genotoxic action, but exhibited cytotoxic, antigenotoxic, and anticytotoxic activities. PMID:26554835

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

PubMed Central

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

PubMed

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Influence of shieldings or antioxidants on DNA damage and early and delyed cell death induced in human fibroblasts by accelerated 595 MeV/u Fe ions

NASA Astrophysics Data System (ADS)

Antonelli, Francesca; Esposito, Giuseppe; Dini, Valentina; Belli, Mauro; Campa, Alessandro; Sorrentino, Eugenio; Antonella Tabocchini, Maria; Lobascio, Cesare; Berra, Bruno

HZE particles from space radiation raise an important protection concern during long-term astronauts' travels. As high charge, high energy particles interact with a shield, both projec-tile and target fragmentation may occurs, so that the biological properties of the emerging radiation field depend on the nature and energy of the incident particles, and on the nature and thickness of the shield. We have studied the influence of PMMA and Kevlar shielding as well as the antioxidant compounds Rosmarinic acid or Resveratrol on DNA damage induction and processing (as evaluated by the g-H2AX phosphorylation assay) and on early and delayed cell death in AG01522 human fibroblasts irradiated with Fe ions of 595 MeV/u at the NASA Space Radiation Laboratory (NSRL), Brookhaven National Laboratory (BNL, Upton, USA). Insertion of PMMA or Kevlar shields (10 g/cm2 thick) gave no substantial change in the bio-logical effect per unit dose on the sample for all the end points studied. When irradiation was performed in the presence of 300 mM Rosmarinic acid or Resveratrol no difference were found for both early and delayed cell death, while a slight protective effect was observed for the initial and residual DNA damage. For both early and delayed cell death, Fe-ions are more effective than g-rays. The number of Fe-ion induced g-H2AX foci is instead lower than that induced by g-rays, due to the presence of multiple DSB within a single focus induced by Fe-ions. From a comparison of the g-H2AX data with the results on DNA fragmentation obtained with 414 MeV/u Fe ions at the Heavy Ions Medical Accelerator (HIMAC, Chiba, Japan) and with 1 GeV/u Fe ions at BNL, in the absence or in the presence of PMMA shields (Esposito et al, Advance in Space Research 2004) we speculate that the overall effect of the shield is a balance between the contributions due to the slowing down of the primary particles and that due to the nuclear fragmentation. Acknowledgment: Financial support from ASI project "From Molecules to Man: Space Re-search Applied to the improvement of the Quality of Life of the Ageing Population on Earth (MoMa)"

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.

Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development.

PubMed

Beltrán-Pardo, Eliana; Jönsson, K Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M; Bernal Villegas, Jaime E

2013-01-01

Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development.

Effects of Ionizing Radiation on Embryos of the Tardigrade Milnesium cf. tardigradum at Different Stages of Development

PubMed Central

Beltrán-Pardo, Eliana; Jönsson, K. Ingemar; Wojcik, Andrzej; Haghdoost, Siamak; Harms-Ringdahl, Mats; Bermúdez-Cruz, Rosa M.; Bernal Villegas, Jaime E.

2013-01-01

Tardigrades represent one of the most desiccation and radiation tolerant animals on Earth, and several studies have documented their tolerance in the adult stage. Studies on tolerance during embryological stages are rare, but differential effects of desiccation and freezing on different developmental stages have been reported, as well as dose-dependent effect of gamma irradiation on tardigrade embryos. Here, we report a study evaluating the tolerance of eggs from the eutardigrade Milnesium cf. tardigradum to three doses of gamma radiation (50, 200 and 500 Gy) at the early, middle, and late stage of development. We found that embryos of the middle and late developmental stages were tolerant to all doses, while eggs in the early developmental stage were tolerant only to a dose of 50 Gy, and showed a declining survival with higher dose. We also observed a delay in development of irradiated eggs, suggesting that periods of DNA repair might have taken place after irradiation induced damage. The delay was independent of dose for eggs irradiated in the middle and late stage, possibly indicating a fixed developmental schedule for repair after induced damage. These results show that the tolerance to radiation in tardigrade eggs changes in the course of their development. The mechanisms behind this pattern are unknown, but may relate to changes in mitotic activities over the embryogenesis and/or to activation of response mechanisms to damaged DNA in the course of development. PMID:24039737

Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi.

PubMed

Pavani, Raphael Souza; da Silva, Marcelo Santos; Fernandes, Carlos Alexandre Henrique; Morini, Flavia Souza; Araujo, Christiane Bezerra; Fontes, Marcos Roberto de Mattos; Sant'Anna, Osvaldo Augusto; Machado, Carlos Renato; Cano, Maria Isabel; Fragoso, Stenio Perdigão; Elias, Maria Carolina

2016-12-01

Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi.

Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

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

Du, Shisuo; Bouquet, Sophie; Lo, Chen-Hao

2015-01-01

Purpose: To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non–small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. Methods and Materials: TGF-β–mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation,more » or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. Results: The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Conclusions: Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.« less

Sulfur mustard analog, 2-chloroethyl ethyl sulfide-induced skin injury involves DNA damage and induction of inflammatory mediators, in part via oxidative stress, in SKH-1 hairless mouse skin.

PubMed

Jain, Anil K; Tewari-Singh, Neera; Gu, Mallikarjuna; Inturi, Swetha; White, Carl W; Agarwal, Rajesh

2011-09-10

Bifunctional alkyalating agent, sulfur mustard (SM)-induced cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 mg or 4 mg CEES for 9-48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and matrix metalloproteinase-9 (MMP-9) levels, indicating the involvement of DNA damage and inflammation in CEES-induced skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-related DNA damage and the induction of inflammatory molecules. Oral GSH (300 mg/kg) administration 1h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injury involves DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injury in humans by SM. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

T7 replisome directly overcomes DNA damage

NASA Astrophysics Data System (ADS)

Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

2015-12-01

Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

Traffic safety for the cell: influence of cyclin-dependent kinase activity on genomic stability.

PubMed

Enders, Greg H; Maude, Shannon L

2006-04-12

Genomic instability has long been considered a key factor in tumorigenesis. Recent evidence suggests that DNA damage may be widespread in early pre-neoplastic states, with deregulation of cyclin-dependent kinase (Cdk) activity a driving force. Increased Cdk activity may critically reduce licensing of origins of DNA replication, drive re-replication, or mediate overexpression of checkpoint proteins, inducing deleterious cell cycle delay. Conversely, inhibition of Cdk activity may compromise replication efficiency, expression of checkpoint proteins, or activation of DNA repair proteins. These vital functions point to the impact of Cdk activity on the stability of the genome. Insight into these pathways may improve our understanding of tumorigenesis and lead to more rational cancer therapies.

Radiation Combined Injury: DNA Damage, Apoptosis, and Autophagy

DTIC Science & Technology

2010-01-01

combined injured-mice, Bacillus and Lactobacillus were isolated within the first 8 d after radiation combined injury. The data imply that mice...Gy followed immediately by 15% total body surface skin-wound trauma. Ileal lysates were prepared 7 d after sham-treatment (Sham), wound- ing (Wound...secretion and increased risk of infection; and ( d ) delayed wound healing—often double the healing time of wounding alone. Since the mechanisms of

Flies, worms and the Free Radical Theory of ageing.

PubMed

Clancy, David; Birdsall, John

2013-01-01

Drosophila and Caenorhabditis elegans have provided the largest body of evidence addressing the Free Radical Theory of ageing, however the evidence has not been unequivocally supportive. Oxidative damage to DNA is probably not a major contributor, damage to lipids is assuming greater importance and damage to proteins probably the source of pathology. On balance the evidence does not support a primary role of oxidative damage in ageing in C. elegans, perhaps because of its particular energy metabolic and stress resistance profile. Evidence is more numerous, varied and consistent and hence more compelling for Drosophila, although not conclusive. However there is good evidence for a role of oxidative damage in later life pathology. Future work should: 1/ make more use of protein oxidative damage measurements; 2/ use inducible transgenic systems or pharmacotherapy to ensure genetic equivalence of controls and avoid confounding effects during development; 3/ to try to delay ageing, target interventions which reduce and/or repair protein oxidative damage. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Toxicity of tributyltin (TBT) to the freshwater planarian Schmidtea mediterranea.

PubMed

Ofoegbu, Pearl U; Simão, Fátima C P; Cruz, Andreia; Mendo, Sónia; Soares, Amadeu M V M; Pestana, João L T

2016-04-01

The freshwater planarian Schmidtea mediterranea, one of the best characterized animal models for regeneration research and developmental biology, is being recognised as a useful species for ecotoxicological studies. Sensitive endpoints related to planarians' behaviour and regeneration can be easily evaluated after exposure to environmental stressors. In this work the sensitivity of S. mediterranea to a gradient of environmentally relevant concentrations of TBT was studied using multiple endpoints like survival, locomotion, head regeneration and DNA damage. In addition, a feeding assay based on planarian's predatory behaviour was performed. Results indicated that TBT is toxic to planarians with LC50's of 1.87 μg L(-1) Sn and 1.31 μg L(-1) Sn at 48 h and 96 h of exposure respectively. Sub-lethal exposures to TBT significantly reduced locomotion and feeding, delayed head regeneration and caused DNA damage in planarians. The behavioural endpoints (feeding and locomotion) and head regeneration were the most sensitive parameters followed by DNA damage. Similar to other aquatic model organisms, S. mediterranea showed high sensitivity towards TBT exposure. Based on our results, and though further research is required concerning their sensitivity to other pollutants, the use of freshwater planarians as a model species in ecotoxicology is discussed. Copyright © 2016. Published by Elsevier Ltd.

Evaluation of cytogenetic and DNA damage in human lymphocytes treated with adrenaline in vitro.

PubMed

Djelić, Ninoslav; Radaković, Milena; Spremo-Potparević, Biljana; Zivković, Lada; Bajić, Vladan; Stevanović, Jevrosima; Stanimirović, Zoran

2015-02-01

Catechol groups can be involved in redox cycling accompanied by generation of reactive oxygen species (ROS) which may lead to oxidative damage of cellular macromolecules including DNA. The objective of this investigation was to evaluate possible genotoxic effects of a natural catecholamine adrenaline in cultured human lymphocytes using cytogenetic (sister chromatid exchange and micronuclei) and the single cell gel electrophoresis (Comet) assay. In cytogenetic tests, six experimental concentrations of adrenaline were used in a range from 0.01-500 μM. There were no indications of genotoxic effects of adrenaline in sister chromatid exchange and micronucleus tests. However, at four highest concentrations of adrenaline (5 μM, 50 μM, 150 μM and 300 μM) we observed a decreased mitotic index and cell-cycle delay. In addition, in the Comet assay we used adrenaline in a range from 0.0005-500 μM, at two treatment times: 15 min or 60 min. In contrast to cytogenetic analysis, there was a dose-dependent increase of DNA damage detected in the Comet assay. These effects were significantly reduced by concomitant treatment with quercetin or catalase. Therefore, the obtained results indicate that adrenaline may exhibit genotoxic effects in cultured human lymphocytes, most likely due to production of reactive oxygen species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Newly identified CHO ERCC3/XPB mutations and phenotype characterization

PubMed Central

Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

2010-01-01

Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596

Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection: no evidence of mitochondrial damage with highly active antiretroviral therapy.

PubMed

Matsukura, Motoi; Chu, Fanny F S; Au, May; Lu, Helen; Chen, Jennifer; Rietkerk, Sonja; Barrios, Rolando; Farley, John D; Montaner, Julio S; Montessori, Valentina C; Walker, David C; Côté, Hélène C F

2008-06-19

Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.

Prevention of DNA damage by L-carnitine induced by metabolites accumulated in maple syrup urine disease in human peripheral leukocytes in vitro.

PubMed

Mescka, Caroline Paula; Wayhs, Carlos Alberto Yasin; Guerreiro, Gilian; Manfredini, Vanusa; Dutra-Filho, Carlos Severo; Vargas, Carmen Regla

2014-09-15

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of L-carnitine (L-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of L-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and L-Car was able to significantly prevent this damage, mainly that due to KIC. Copyright © 2014 Elsevier B.V. All rights reserved.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

PubMed

Vermeij, W P; Dollé, M E T; Reiling, E; Jaarsma, D; Payan-Gomez, C; Bombardieri, C R; Wu, H; Roks, A J M; Botter, S M; van der Eerden, B C; Youssef, S A; Kuiper, R V; Nagarajah, B; van Oostrom, C T; Brandt, R M C; Barnhoorn, S; Imholz, S; Pennings, J L A; de Bruin, A; Gyenis, Á; Pothof, J; Vijg, J; van Steeg, H; Hoeijmakers, J H J

2016-09-15

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1 ∆/- ) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg -/- (also known as Ercc5 -/- ) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1 ∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1 ∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1 ∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

EdU induces DNA damage response and cell death in mESC in culture.

PubMed

Kohlmeier, Fanni; Maya-Mendoza, Apolinar; Jackson, Dean A

2013-03-01

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

PubMed Central

Pavani, Raphael Souza; da Silva, Marcelo Santos; Fernandes, Carlos Alexandre Henrique; Morini, Flavia Souza; Araujo, Christiane Bezerra; Fontes, Marcos Roberto de Mattos; Sant’Anna, Osvaldo Augusto; Machado, Carlos Renato; Cano, Maria Isabel; Fragoso, Stenio Perdigão; Elias, Maria Carolina

2016-01-01

Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi. PMID:27984589

PCNA-dependent accumulation of CDKN1A into nuclear foci after ionizing irradiation

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

Wiese, Claudia; Rudolph, Jeanette Heede; Jakob, Burkhard

2012-03-26

The cyclin-dependent kinase inhibitor CDKN1A/p21 confers cell-cycle arrest in response to DNA damage and inhibits DNA replication through its direct interaction with the proliferating cell nuclear antigen (PCNA) and cyclin/cyclin-dependent kinase complexes. Previously, we reported that in response to densely ionizing radiation CDKN1A rapidly is recruited to the sites of particle traversal, and that CDKN1A foci formation in response to heavy ions is independent of its transactivation by TP53. In this paper, we show that exposure of normal human fibroblasts to X-rays or to H 2O 2 also induces nuclear accumulations of CDKN1A. We find that CDKN1A foci formation inmore » response to radiation damage is dependent on its dephosphorylation and on its direct physical interaction with PCNA. Live cell imaging analyses of ectopically expressed EGFP-CDKN1A and dsRed-PCNA show rapid recruitment of both proteins into foci after radiation damage. Detailed dynamic measurements reveal a slightly delayed recruitment of CDKN1A compared to PCNA, which is best described by bi-exponential curve fitting, taking the preceding binding of PCNA to DNA into account. Finally, we propose a regulatory role for CDKN1A in mediating PCNA function after radiation damage, and provide evidence that this role is distinct from its involvement in nucleotide excision repair and unrelated to double-strand break repair.« less

Sulforaphane inhibits damage-induced poly (ADP-ribosyl)ation via direct interaction of its cellular metabolites with PARP-1.

PubMed

Piberger, Ann Liza; Keil, Claudia; Platz, Stefanie; Rohn, Sascha; Hartwig, Andrea

2015-11-01

The isothiocyanate sulforaphane, a major breakdown product of the broccoli glucosinolate glucoraphanin, has frequently been proposed to exert anticarcinogenic properties. Potential underlying mechanisms include a zinc release from Kelch-like ECH-associated protein 1 followed by the induction of detoxifying enzymes. This suggests that sulforaphane may also interfere with other zinc-binding proteins, e.g. those essential for DNA repair. Therefore, we explored the impact of sulforaphane on poly (ADP-ribose)polymerase-1 (PARP-1), poly (ADP-ribosyl)ation (PARylation), and DNA single-strand break repair (SSBR) in cell culture. Immunofluorescence analyses showed that sulforaphane diminished H2 O2 -induced PARylation in HeLa S3 cells starting from 15 μM despite increased lesion induction under these conditions. Subcellular experiments quantifying the damage-induced incorporation of (32) P-ADP-ribose by PARP-1 displayed no direct impact of sulforaphane itself, but cellular metabolites, namely the glutathione conjugates of sulforaphane and its interconversion product erucin, reduced PARP-1 activity concentration dependently. Interestingly, this sulforaphane metabolite-induced PARP-1 inhibition was prevented by thiol compounds. PARP-1 is a stimulating factor for DNA SSBR-rate and we further demonstrated that 25 μM sulforaphane also delayed the rejoining of H2 O2 -induced DNA strand breaks, although this might be partly due to increased lesion frequencies. Sulforaphane interferes with damage-induced PARylation and SSBR, which implies a sulforaphane-dependent impairment of genomic stability. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epigenetic perturbations in the pathogenesis of mustard toxicity; hypothesis and preliminary results

PubMed Central

Korkmaz, Ahmet; Yaren, Hakan; Kunak, Z. Ilker; Uysal, Bulent; Kurt, Bulent; Topal, Turgut; Kenar, Levent; Ucar, Ergun; Oter, Sukru

2008-01-01

Among the most readily available chemical warfare agents, sulfur mustard (SM), also known as mustard gas, has been the most widely used chemical weapon. SM causes debilitating effects that can leave an exposed individual incapacitated for days to months; therefore delayed SM toxicity is of much greater importance than its ability to cause lethality. Although not fully understood, acute toxicity of SM is related to reactive oxygen and nitrogen species, oxidative stress, DNA damage, poly(ADP-ribose) polymerase (PARP) activation and energy depletion within the affected cell. Therefore several antioxidants and PARP inhibitors show beneficial effects against acute SM toxicity. The delayed toxicity of SM however, currently has no clear mechanistic explanation. One third of the 100,000 Iranian casualties are still suffering from the detrimental effects of SM in spite of the extensive treatment. We, therefore, made an attempt whether epigenetic aberrations may contribute to pathogenesis of mustard poisoning. Preliminary evidence reveals that mechlorethamine (a nitrogen mustard derivative) exposure may not only cause oxidative stress, DNA damage, but epigenetic perturbations as well. Epigenetic refers to the study of changes that influence the phenotype without causing alteration of the genotype. It involves changes in the properties of a cell that are inherited but do not involve a change in DNA sequence. It is now known that in addition to mutations, epimutations contribute to a variety of human diseases. Under light of preliminary results, the current hypothesis will focus on epigenetic regulations to clarify mustard toxicity and the use of drugs to correct possible epigenetic defects. PMID:21218122

Cells-nano interactions and molecular toxicity after delayed hypersensitivity, in guinea pigs on exposure to hydroxyapatite nanoparticles.

PubMed

Geetha, C S; Remya, N S; Leji, K B; Syama, S; Reshma, S C; Sreekanth, P J; Varma, H K; Mohanan, P V

2013-12-01

The aim of the study was to evaluate the cells-nanoparticle interactions and molecular toxicity after delayed hypersensitivity in Guinea pigs, exposed to hydroxyapatite nanoparticles (HANP). The study focuses on synthesizing and characterizing HANPs and gaining an insight into the cytotoxicity, molecular toxicity, hypersensitivity and oxidative stress caused by them in vitro and in vivo. HANP was synthesized by chemical method and characterized by standard methods. Cytotoxicity was assessed on L929 cells by MTT assay and in vitro studies were carried out on rat liver homogenate. In vivo study was carried out by topical exposure of Guinea pigs with HANP, repeatedly, and evaluating the skin sensitization potential, blood parameters, oxidative stress in liver and brain and DNA damage (8-hydroxyl-2-deoxyguanosine: 8-OHdG) in liver. The results of the study indicated that there was no cytotoxicity (up to 600μg/mL) and oxidative damage (up to 100μg/mL), when exposed to HANPs. It was also evident that, there was no skin sensitization and oxidative damage when HANP were exposed to Guinea pigs. Copyright © 2013 Elsevier B.V. All rights reserved.

Delayed effects of low level acute irradiation and chronic environmental radioactive contamination on DNA lymphocytes of people living in Dolon, a settlement located in the vicinity of the Semipalatinsk nuclear test site (Kazakhstan).

PubMed

Chenal, C; Legue, F; Nourgalieva, K

2006-10-01

During 42 years several hundred nuclear tests were performed by the former USSR at the Semipalatinsk Test Site (STS, Kazakhstan), of which more than 100 were done in the atmosphere. We report here the late genetic damage of external exposure to radiation and environmental radioactive contamination in people living in Dolon, a small settlement situated in the vicinity of the STS. The comet assay was applied on DNA lymphocytes of 20 exposed women and 32 non-exposed women living at 500 km from the STS. We observed a statistically significant difference between the exposed and control groups for mean tail moment (MTM) and DNA% in the tail. The mean values of all comet assay parameters (MTM, DNA% in the tail and score) were higher in the group of women born before 1949 as compared to those born after 1950, which could reflect an effect of external irradiation in 1949 due to the most contaminating explosion. These results suggest that people exposed 50 years ago to relatively small doses of external irradiation and/or still living in an environment contaminated by small amounts of long life radionuclides, still present DNA damage which is in agreement with other cytogenetical studies performed at the same site, on the same population.

Effects of delay and noise in a negative feedback regulatory motif

NASA Astrophysics Data System (ADS)

Palassini, Matteo; Dies, Marta

2009-03-01

The small copy number of the molecules involved in gene regulation can induce nontrivial stochastic phenomena such as noise-induced oscillations. An often neglected aspect of regulation dynamics are the delays involved in transcription and translation. Delays introduce analytical and computational complications because the dynamics is non-Markovian. We study the interplay of noise and delays in a negative feedback model of the p53 core regulatory network. Recent experiments have found pronounced oscillations in the concentrations of proteins p53 and Mdm2 in individual cells subjected to DNA damage. Similar oscillations occur in the Hes-1 and NK-kB systems, and in circadian rhythms. Several mechanisms have been proposed to explain this oscillatory behaviour, such as deterministic limit cycles, with and without delay, or noise-induced excursions in excitable models. We consider a generic delayed Master Equation incorporating the activation of Mdm2 by p53 and the Mdm2-promoted degradation of p53. In the deterministic limit and for large delays, the model shows a Hopf bifurcation. Via exact stochastic simulations, we find strong noise-induced oscillations well outside the limit-cycle region. We propose that this may be a generic mechanism for oscillations in gene regulatory systems.

Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override

PubMed Central

Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

2014-01-01

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25. PMID:24666325

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

PubMed

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-07-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster

PubMed Central

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-01-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390

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

Anthocyanins from Black Chokeberry (Aroniamelanocarpa Elliot) Delayed Aging-Related Degenerative Changes of Brain.

PubMed

Wei, Jie; Zhang, Guokun; Zhang, Xiao; Xu, Dexin; Gao, Jun; Fan, Jungang; Zhou, Zhiquan

2017-07-26

Aging is the greatest risk factor for most neurodegenerative diseases, which is associated with decreasing cognitive function and significantly affecting life quality in the elderly. Computational analysis suggested that 4 anthocyanins from chokeberry fruit increased Klotho (aging-suppressor) structural stability, so we hypothesized that chokeberry anthocyanins could antiaging. To explore the effects of anthocyanins treatment on brain aging, mice treated with 15 or 30 mg/kg anthocyanins by gavage and injected D-galactose accelerated aging per day. After 8 weeks, cognitive and noncognitive components of behavior were determined. Our studies showed that anthocyanins blocked age-associated cognitive decline and response capacity in senescence accelerated mice. Furthermore, mice treated with anthocyanins-supplemented showed better balance of redox systems (SOD, GSH-PX, and MDA) in all age tests. Three major monoamines were norepinephrine, dopamine, and 5-hydroxytryptamine, and their levels were significantly increased; the levels of inflammatory cytokines (COX2, TGF-β1, and IL-1) transcription and DNA damage were decreased significantly in brains of anthocyanins treated mice compared to aged models. The DNA damage signaling pathway was also regulated with anthocyanins. Our results suggested that anthocyanins was a potential approach for maintaining thinking and memory in aging mice, possibly by regulating the balance of redox system and reducing inflammation accumulation, and the most important factor was inhibiting DNA damage.

Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma

PubMed Central

Zeng, Ling; Beggs, Reena R.; Cooper, Tiffiny S.; N.Weaver, Alice; S.Yang, Eddy

2017-01-01

EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo, suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644). PMID:28138028

Brief report: a human induced pluripotent stem cell model of cernunnos deficiency reveals an important role for XLF in the survival of the primitive hematopoietic progenitors.

PubMed

Tilgner, Katarzyna; Neganova, Irina; Singhapol, Chatchawan; Saretzki, Gabriele; Al-Aama, Jumana Yousuf; Evans, Jerome; Gorbunova, Vera; Gennery, Andrew; Przyborski, Stefan; Stojkovic, Miodrag; Armstrong, Lyle; Jeggo, Penny; Lako, Majlinda

2013-09-01

Cernunnos (also known as XLF) deficiency syndrome is a rare recessive autosomal disorder caused by mutations in the XLF gene, a key factor involved in the end joining step of DNA during nonhomologous end joining (NHEJ) process. Human patients with XLF mutations display microcephaly, developmental and growth delays, and severe immunodeficiency. While the clinical phenotype of DNA damage disorders, including XLF Syndrome, has been described extensively, the underlying mechanisms of disease onset, are as yet, undefined. We have been able to generate an induced pluripotent stem cell (iPSC) model of XLF deficiency, which accurately replicates the double-strand break repair deficiency observed in XLF patients. XLF patient-specific iPSCs (XLF-iPSC) show typical expression of pluripotency markers, but have altered in vitro differentiation capacity and an inability to generate teratomas comprised of all three germ layers in vivo. Our results demonstrate that XLF-iPSCs possess a weak NHEJ-mediated DNA repair capacity that is incapable of coping with the DNA lesions introduced by physiological stress, normal metabolism, and ionizing radiation. XLF-iPSC lines are capable of hematopoietic differentiation; however, the more primitive subsets of hematopoietic progenitors display increased apoptosis in culture and an inability to repair DNA damage. Together, our findings highlight the importance of NHEJ-mediated-DNA repair in the maintenance of a pristine pool of hematopoietic progenitors during human embryonic development. © AlphaMed Press.

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

PubMed

Rissel, D; Losch, J; Peiter, E

2014-11-01

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Persistence of Repair Proteins at Unrepaired DNA Damage Distinguishes Diseases with ERCC2 (XPD) Mutations: Cancer-Prone Xeroderma Pigmentosum vs. Non-Cancer-Prone Trichothiodystrophy

PubMed Central

Boyle, Jennifer; Ueda, Takahiro; Oh, Kyu-Seon; Imoto, Kyoko; Tamura, Deborah; Jagdeo, Jared; Khan, Sikandar G.; Nadem, Carine; DiGiovanna, John J.; Kraemer, Kenneth H.

2012-01-01

Patients with xeroderma pigmentosum (XP) have a 1,000-fold increase in ultraviolet (UV)-induced skin cancers while trichothiodystrophy (TTD) patients, despite mutations in the same genes, ERCC2 (XPD) or ERCC3 (XPB), are cancer-free. Unlike XP cells, TTD cells have a nearly normal rate of removal of UV-induced 6-4 photoproducts (6-4PP) in their DNA and low levels of the basal transcription factor, TFIIH. We examined seven XP, TTD, and XP/TTD complex patients and identified mutations in the XPD gene. We discovered large differences in nucleotide excision repair (NER) protein recruitment to sites of localized UV damage in TTD cells compared to XP or normal cells. XPC protein was rapidly localized in all cells. XPC was redistributed in TTD, and normal cells by 3 hr postirradiation, but remained localized in XP cells at 24-hr postirradiation. In XP cells recruitment of other NER proteins (XPB, XPD, XPG, XPA, and XPF) was also delayed and persisted at 24 hr (p < 0.001). In TTD cells with defects in the XPD, XPB, or GTF2H5 (TTDA) genes, in contrast, recruitment of these NER proteins was reduced compared to normals at early time points (p < 0.001) and remained low at 24 hr postirradiation. These data indicate that in XP persistence of NER proteins at sites of unrepaired DNA damage is associated with greatly increased skin cancer risk possibly by blockage of translesion DNA synthesis. In contrast, in TTD, low levels of unstable TFIIH proteins do not accumulate at sites of unrepaired photoproducts and may permit normal translesion DNA synthesis without increased skin cancer. PMID:18470933

Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells.

PubMed

Haendeler, Judith; Hoffmann, Jörg; Diehl, J Florian; Vasa, Mariuca; Spyridopoulos, Ioakim; Zeiher, Andreas M; Dimmeler, Stefanie

2004-04-02

Aging is associated with a rise in intracellular reactive oxygen species (ROS) and a loss of telomerase reverse transcriptase activity. Incubation with H2O2 induced the nuclear export of telomerase reverse transcriptase (TERT) into the cytosol in a Src-family kinase-dependent manner. Therefore, we investigated the hypothesis that age-related increase in reactive oxygen species (ROS) may induce the nuclear export of TERT and contribute to endothelial cell senescence. Continuous cultivation of endothelial cells resulted in an increased endogenous formation of ROS starting after 29 population doublings (PDL). This increase was accompanied by mitochondrial DNA damage and preceded the onset of replicative senescence at PDL 37. Along with the enhanced formation of ROS, we detected an export of nuclear TERT protein from the nucleus into the cytoplasm and an activation of the Src-kinase. Moreover, the induction of premature senescence by low concentrations of H2O2 was completely blocked with the Src-family kinase inhibitor PP2, suggesting a crucial role for Src-family kinases in the induction of endothelial cell aging. Incubation with the antioxidant N-acetylcysteine, from PDL 26, reduced the intracellular ROS formation and prevented mitochondrial DNA damage. Likewise, nuclear export of TERT protein, loss in the overall TERT activity, and the onset of replicative senescence were delayed by incubation with N-acetylcysteine. Low doses of the statin, atorvastatin (0.1 micromol/L), had also effects similar to those of N-acetylcysteine. We conclude that both antioxidants and statins can delay the onset of replicative senescence by counteracting the increased ROS production linked to aging of endothelial 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.

Direct non transcriptional role of NF-Y in DNA replication.

PubMed

Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

2016-04-01

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Impact of Charged Particle Exposure on Homologous DNA Double-Strand Break Repair in Human Blood-Derived Cells

PubMed Central

Rall, Melanie; Kraft, Daniela; Volcic, Meta; Cucu, Aljona; Nasonova, Elena; Taucher-Scholz, Gisela; Bönig, Halvard; Wiesmüller, Lisa; Fournier, Claudia

2015-01-01

Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment. PMID:26618143

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 Seed Repair Response during Germination: Disclosing Correlations between DNA Repair, Antioxidant Response, and Chromatin Remodeling in Medicago truncatula

PubMed Central

Pagano, Andrea; Araújo, Susana de Sousa; Macovei, Anca; Leonetti, Paola; Balestrazzi, Alma

2017-01-01

This work provides novel insights into the effects caused by the histone deacetylase inhibitor trichostatin A (TSA) during Medicago truncatula seed germination, with emphasis on the seed repair response. Seeds treated with H2O and TSA (10 and 20 μM) were collected during imbibition (8 h) and at the radicle protrusion phase. Biometric data showed delayed germination and impaired seedling growth in TSA-treated samples. Comet assay, performed on radicles at the protrusion phase and 4-days old M. truncatula seedlings, revealed accumulation of DNA strand breaks upon exposure to TSA. Activation of DNA repair toward TSA-mediated genotoxic damage was evidenced by the up-regulation of MtOGG1(8-OXOGUANINE GLYCOSYLASE/LYASE) gene involved in the removal of oxidative DNA lesions, MtLIGIV(LIGASE IV) gene, a key determinant of seed quality, required for the rejoining of DNA double strand breaks and TDP(TYROSYL-DNA PHOSPHODIESTERASE) genes encoding the multipurpose DNA repair enzymes tyrosyl-DNA phosphodiesterases. Since radical scavenging can prevent DNA damage, the specific antioxidant activity (SAA) was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu reagent assays. Fluctuations of SAA were observed in TSA-treated seeds/seedlings concomitant with the up-regulation of antioxidant genes MtSOD(SUPEROXIDE DISMUTASE, MtAPX(ASCORBATE PEROXIDASE) and MtMT2(TYPE 2 METALLOTHIONEIN). Chromatin remodeling, required to facilitate the access of DNA repair enzymes at the damaged sites, is also part of the multifaceted seed repair response. To address this aspect, still poorly explored in plants, the MtTRRAP(TRANSFORMATION/TRANSACTIVATION DOMAIN-ASSOCIATED PROTEIN) gene was analyzed. TRRAP is a transcriptional adaptor, so far characterized only in human cells where it is needed for the recruitment of histone acetyltransferase complexes to chromatin during DNA repair. The MtTRRAP gene and the predicted interacting partners MtHAM2 (HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY) and MtADA2A (TRANSCRIPTIONAL ADAPTOR) showed tissue- and dose-dependent fluctuations in transcript levels. PCA (Principal Component Analysis) and correlation analyses suggest for a new putative link between DNA repair and chromatin remodeling that involves MtOGG1 and MtTRRAP genes, in the context of seed germination. Interesting correlations also connect DNA repair and chromatin remodeling with antioxidant players and proliferation markers. PMID:29184569

Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase.

PubMed

Tanaka, Miyuki; Takahara, Michiyo; Nukina, Kohei; Hayashi, Akiyo; Sakai, Wataru; Sugasawa, Kaoru; Shiomi, Yasushi; Nishitani, Hideo

2017-04-03

Cdt1 is rapidly degraded by CRL4 Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4 Cdt2 . The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4 Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

The radiation response of human dermal fibroblasts

NASA Astrophysics Data System (ADS)

Mitchell, Stephen Andrew

A clinically reliable predictive assay based on normal-tissue radiosensitivity may lead to improved tumour control through individualised dose prescriptions. In-vitro fibroblast radiosensitivity has been shown, in several studies, to correlate with late radiation morbidity. The aim of this study was to investigate some of the cellular mechanisms underlying the normal-tissue response. In this study, seventeen primary fibroblast strains were established by enzymatic disaggregation of skin biopsies obtained from patients. These comprised seven who experienced acute tissue reactions to radiotherapy, four patients with a normal response and six non-cancer volunteers. An AT cell line was included as a positive control for radiosensitivity. In-vitro radiosensitivity was measured using a clonogenic assay at both high (HDR: 1.6 Gymin-1) and low dose rate (LDR: 0.01 Gymin-1). The radiation parameter HDR SF2 was the most sensitive in discriminating the seven sensitive patients from the remaining ten normal patients (range 0.11-0.19 sensitive patients compared with 0.17-0.34 control patients: p<0.0001). Neither the use of an internal control or LDR radiation protocol increased this discrimination. Pulsed-field gel electrophoresis (PFGE) was used to measure the level of initial and residual double-strand breaks following irradiation. No correlation was found between HDR SF2 and initial DNA damage. However, a strong correlation was found between clonogenic survival and both residual DNA damage (measured over 10-70 Gy, allowing 4 h repair, correlation coefficient: 0.90, <0.0001) and the ratio of residual/initial DNA damage, with the sensitive cell lines generally showing a higher level of residual DNA damage. Cell-cycle delays were found in all 18 cell strains in response to 2 Gy irradiation, but were not found to discriminate between sensitive and normal patients. Associated studies found no mutations of the ATM gene in the five radiosensitive patients studied. However, a coding sequence alteration was found in the XRCC1 gene in one of the radiosensitive patients. These findings indicate that a DNA repair defect may be partly responsible for the extreme reactions to radiotherapy observed in a small percentage of patients and that with further modifications, an assay based on measurement of residual DNA damage may form the basis of a predictive test for radiosensitivity.

The role of glutathione in DNA damage by potassium bromate in vitro.

PubMed

Parsons, J L; Chipman, J K

2000-07-01

We have investigated the role of reduced glutathione (GSH) in the genetic toxicity of the rodent renal carcinogen potassium bromate (KBrO(3)). A statistically significant increase in the concentration of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine was measured following incubation of calf thymus DNA with KBrO(3) and GSH or N-acetylcysteine (NACys). This was dependent on these thiols and was associated with the loss of GSH and production of oxidized glutathione. A short-lived (<6 min) intermediate was apparent which did not react with the spin trap dimethylpyrroline N-oxide. DNA oxidation was not evident when potassium chlorate (KClO(3)) or potassium iodate (KIO(3)) were used instead of KBrO(3), though GSH depletion also occurred with KIO(3), but not with KClO(3). Other reductants and thiols in combination with KBrO(3) did not cause a significant increase in DNA oxidation. DNA strand breakage was also induced by KBrO(3) in human white blood cells (5 mM) and rat kidney epithelial cells (NRK-52E, 1.5 mM). This was associated with an apparent small depletion of thiols in NRK-52E cells at 15 min and with an elevation of 8-oxodG at a delayed time of 24 h. Depletion of intra-cellular GSH by diethylmaleate in human lymphocytes decreased the amount of strand breakage induced by KBrO(3). Extracellular GSH, however, protected against DNA strand breakage by KBrO(3), possibly due to the inability of the reactive product to enter the cell. In contrast, membrane-permeant NACys enhanced KBrO(3)-induced DNA strand breakage in these cells. DNA damage by KBrO(3) is therefore largely dependent on access to intracellular GSH.

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.

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.

PubMed

Ahmed, Emad A; Vélaz, Eukene; Rosemann, Michael; Gilbertz, Klaus-P; Scherthan, Harry

2017-03-01

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G 1 -like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

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.

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.

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.

DNA damage in oral cancer and normal cells induced by nitrogen atmospheric pressure plasma jets

NASA Astrophysics Data System (ADS)

Han, Xu; Kapaldo, James; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

2015-09-01

Nitrogen atmospheric pressure plasma jets (APPJs) have been shown to effectively induce DNA double strand breaks in SCC25 oral cancer cells. The APPJ source constructed in our laboratory operates based on dielectric barrier discharge. It consists of two copper electrodes alternatively wrapping around a fused silica tube with nitrogen as a feed gas. It is generally more challenging to ignite plasma in N2 atmosphere than in noble gases. However, N2 provides additional advantages such as lower costs compared to noble gases, thus this design can be beneficial for the future long-term clinical use. To compare the effects of plasma on cancer cells (SCC25) and normal cells (OKF), the cells from both types were treated at the same experimental condition for various treatment times. The effective area with different damage levels after the treatment was visualized as 3D maps. The delayed damage effects were also explored by varying the incubation times after the treatment. All of these studies are critical for a better understanding of the damage responses of cellular systems exposed to the plasma radiation, thus are useful for the development of the advanced plasma cancer therapy. The research described herein was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Basic Energy Sciences, Office of Science, United States Department of Energy through Grant No. DE-FC02-04ER15533.

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...

The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

PubMed

Du, W; Amarachintha, S; Wilson, A; Pang, Q

2017-02-01

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca -/- hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca -/- pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca -/- pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca -/- pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

Radiosensitivity and Induction of Apoptosis by High LET Carbon Ion Beam and Low LET Gamma Radiation: A Comparative Study

PubMed Central

Ghorai, Atanu; Bhattacharyya, Nitai P.; Sarma, Asitikantha; Ghosh, Utpal

2014-01-01

Cancer treatment with high LET heavy ion beam, especially, carbon ion beam (12C), is becoming very popular over conventional radiotherapy like low LET gamma or X-ray. Combination of Poly(ADP-ribose) polymerase (PARP) inhibitor with xenotoxic drugs or conventional radiation (gamma or X-ray) is the newer approach for cancer therapy. The aim of our study was to compare the radiosensitivity and induction of apoptosis by high LET 12C and low LET gamma radiation in HeLa and PARP-1 knocked down cells. We did comet assay to detect DNA breaks, clonogenic survival assay, and cell cycle analysis to measure recovery after DNA damage. We measured apoptotic parameters like nuclear fragmentation and caspase-3 activation. DNA damage, cell killing, and induction of apoptosis were significantly higher for 12C than gamma radiation in HeLa. Cell killing and apoptosis were further elevated upon knocking down of PARP-1. Both 12C and gamma induced G2/M arrest although the 12C had greater effect. Unlike the gamma, 12C irradiation affects DNA replication as detected by S-phase delay in cell cycle analysis. So, we conclude that high LET 12C has greater potential over low LET gamma radiation in killing cells and radiosensitization upon PARP-1 inhibition was several folds greater for 12C than gamma. PMID:25018892

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

Anti-aging Effect of Transplanted Amniotic Membrane Mesenchymal Stem Cells in a Premature Aging Model of Bmi-1 Deficiency

PubMed Central

Xie, Chunfeng; Jin, Jianliang; Lv, Xianhui; Tao, Jianguo; Wang, Rong; Miao, Dengshun

2015-01-01

To determine whether transplanted amniotic membrane mesenchymal stem cells (AMSCs) ameliorated the premature senescent phenotype of Bmi-1-deficient mice, postnatal 2-day-old Bmi-1−/− mice were injected intraperitoneally with the second-passage AMSCs from amniotic membranes of β-galactosidase (β-gal) transgenic mice or wild-type (WT) mice labeled with DiI. Three reinjections were given, once every seven days. Phenotypes of 5-week-old β-gal+ AMSC-transplanted or 6-week-old DiI+ AMSC-transplanted Bmi-1−/− mice were compared with vehicle-transplanted Bmi-1−/− and WT mice. Vehicle-transplanted Bmi-1−/− mice displayed growth retardation and premature aging with decreased cell proliferation and increased cell apoptosis; a decreased ratio and dysmaturity of lymphocytic series; premature osteoporosis with reduced osteogenesis and increased adipogenesis; redox imbalance and DNA damage in multiple organs. Transplanted AMSCs carried Bmi-1 migrated into multiple organs, proliferated and differentiated into multiple tissue cells, promoted growth and delayed senescence in Bmi-1−/− transplant recipients. The dysmaturity of lymphocytic series were ameliorated, premature osteoporosis were rescued by promoting osteogenesis and inhibiting adipogenesis, the oxidative stress and DNA damage in multiple organs were inhibited by the AMSC transplantation in Bmi-1−/− mice. These findings indicate that AMSC transplantation ameliorated the premature senescent phenotype of Bmi-1-deficient mice and could be a novel therapy to delay aging and prevent aging-associated degenerative diseases. PMID:26370922

Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma.

PubMed

Zeng, Ling; Beggs, Reena R; Cooper, Tiffiny S; Weaver, Alice N; Yang, Eddy S

2017-04-01

EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo , suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644). Mol Cancer Ther; 16(4); 591-600. ©2017 AACR . ©2017 American Association for Cancer Research.

Effect of modified wuzi yanzong granule on patients with mild cognitive impairment from oxidative damage aspect.

PubMed

Wang, Xue-mei; Fu, Hong; Liu, Geng-xin; Zhu, Wei; Li, Li; Yang, Jin-xia

2007-12-01

To observe the effects of modified Wuzi Yanzong Granule (WYG) on memory function and the activity of serum superoxide dismutase (SOD), malondialdehyde (MDA) levels, leukocyte mitochondrial DNA (mtDNA) deletion rate and beta-amyloid protein(1-28) (A beta(1-28)) in patients with mild cognitive impairment (MCI). Thirty-six patients with MCI were selected based on the internationally recognized Petersen's criteria, and equally and randomly assigned to two groups. The treated group was treated with WYG and the control group was treated with placebo for 3 months. In addition, 20 healthy subjects were included in the study as the normal control group. Changes of memory function, SOD activity, MDA content, leukocyte mtDNA deletion rate and A beta(1-28) content were observed before and after treatment. Compared with the normal control group, the memory quotient and SOD activity in patients with MCI decreased significantly (P < 0.01), while MDA, A beta(1-28) levels and the leukocyte mtDNA deletion rate increased significantly (P < 0.01). After treatment, levels of memory quotient and serum SOD activity increased while the serum MDA level, leukocyte mtDNA deletion rate and A beta(1-28) level decreased in the treated group compared with those before treatment (P<0.01, P<0.05). Meanwhile, leukocyte mtDNA deletion rate and A beta(1-28) content in the treated group were all lower than those in the control group (P<0.05). WYG could improve memory function in patients with MCI and the therapeutic mechanism is possibly related to the increased activity of anti-oxidase, the improved free radical metabolism and the alleviation of leukocyte mtDNA oxidation damage. WYG shows clinical significance in delaying the progression of MCI.

Enhanced anticancer effects of a mixture of low-dose mushrooms and Panax ginseng root extracts in human colorectal cancer cells.

PubMed

Lee, Mi So; Kim, Mi-Sook; Yoo, Jae Kuk; Lee, Ji Young; Ju, Jae Eun; Jeong, Youn Kyoung

2017-09-01

Worldwide, colorectal cancer is the third most common cancer in men and the second most common in women. As conventional colorectal cancer therapies result in various side effects, there is a need for adjuvant therapy that can enhance the conventional therapies without complications. In this study, we investigated the anticancer effects of combined mixture of the several medicinal mushrooms and Panax ginseng root extracts (also called Amex7) as an adjuvant compound in the treatment of human colorectal cancer. We observed the in vivo inhibitory effect of Amex7 (1.25, 6.25, and 12.5 ml/kg, oral administration, twice daily) on tumor growth in a mouse model xenografted with HT-29 human colorectal cancer cells. In vitro, at 6, 12, and 24 h after 4% Amex7 treatment, we analyzed cell cycle by flow cytometry and the expression levels of cell cycle progression, apoptosis, and DNA damage repair-related proteins using immunoblotting and immunofluorescence staining in HT-29 cell line. As a result, Amex7 significantly suppressed tumor growth in HT-29 human colorectal cancer cells and xenografts. In vitro, Amex7 induced G2/M arrest through the regulation of cell cycle proteins and cell death by apoptosis and autophagy. Additionally, Amex7 consistently induced DNA damage and delayed the repair of Amex7-induced DNA damage by reducing the level of HR repair proteins. In conclusion, Amex7 enhanced anticancer effects through the induction of G2/M arrest and cell death, including apoptosis and autophagy. Furthermore, Amex7 impaired DNA damage repair. The present study provides a scientific rationale for the clinical use of a combined mixture of medicinal mushrooms and P. ginseng root extracts as an adjuvant treatment in human colorectal cancer.

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

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

Wei, Ren-Jie

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosismore » in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells. • ABT-751 induces early autophagy and delays apoptosis. • ABT-751 induces autophagy via modulations of nuclear TP53 and AKT/MTOR pathways. • ABT-751-induced apoptosis is ROS-, mitochondria- and caspase-dependent. • Inhibition of autophagy enhances ABT-751-induced apoptosis.« less

Cellular and molecular effects of the liposomal mTHPC derivative Foslipos in prostate carcinoma cells in vitro.

PubMed

Gyenge, Emina Besic; Hiestand, Seraina; Graefe, Susanna; Walt, Heinrich; Maake, Caroline

2011-06-01

Meso-tetra-hydroxyphenyl-chlorine (mTHPC) is among the most powerful photosensitizers available for photodynamic therapy (PDT). However, the mechanisms leading to cell death are poorly understood. We here focused on changes at DNA and RNA levels after treatment with the liposomal mTHPC derivative Foslipos in vitro. After determination of darktoxicity, laser conditions and uptake kinetics, PC-3 prostate carcinoma cells were subjected to PDT with Foslipos, followed by assessment of cell numbers directly (TP0) or 1h (TP1), 2h (TP2), 5h (TP5) and 24h (TP24) after illumination. Nucleic acids had been extracted for evaluation of RNA amounts and integrity as well as for estimation of abasic sites as a measure for DNA damage. Furthermore, expression changes of 84 genes related to oxidative stress were investigated by quantitative polymerase chain reaction. Already at TP0, the number of dead cells was significantly higher after PDT versus controls and at TP24 more than 90% of cells had been destroyed. PDT resulted in a severe damage of both RNA and DNA. Gene expression analyses revealed an impact of PDT on pathways for oxidative and metabolic stress, heat shock, proliferation and carcinogenesis, growth arrest, inflammation, DNA repair and apoptosis signaling. Mechanisms of Foslipos-mediated PDT comprise a combination of acute and delayed lethal effects in PC-3 cells. The latter may include death processes initiated by nucleic acid damage, activation of stress and growth arrest genes in combination with a reduced capability to adequately cope with oxidative toxicity. Our results will help to better understand molecular photodynamic effects. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Decitabine induces delayed reactive oxygen species (ROS) accumulation in leukemia cells and induces the expression of ROS generating enzymes.

PubMed

Fandy, Tamer E; Jiemjit, Anchalee; Thakar, Manjusha; Rhoden, Paulette; Suarez, Lauren; Gore, Steven D

2014-03-01

Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently approved by the U.S. Food and Drug Administration for treatment of myelodysplastic syndrome. The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation. Flow cytometry was used for cell cycle, apoptosis, and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using real-time PCR. DNA methylation was detected by methylation-specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis. 5-Aza-2'-deoxycytidine (DAC) induced cell-cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation. These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC. ©2014 AACR

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins.

PubMed

Maachani, Uday Bhanu; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J; Camphausen, Kevin; Tandle, Anita

2015-05-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. ©2015 American Association for Cancer Research.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins

PubMed Central

Maachani, Uday B.; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J.; Camphausen, Kevin; Tandle, Anita

2015-01-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells was analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor, NMS-P715 on radiosensitivity in multiple model systems including: GBM cell lines, a normal astrocyte and a normal fibroblast cell line. DNA double strand breaks (DSBs) were evaluated using γH2AX foci and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Further, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of post-radiation mitotic catastrophe. MNS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1 silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair and replication including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. PMID:25722303

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

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.

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.

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.

XERODERMA PIGMENTOSUM, TRICHOTHIODYSTROPHY AND COCKAYNE SYNDROME: A COMPLEX GENOTYPE-PHENOTYPE RELATIONSHIP

PubMed Central

Kraemer, Kenneth H.; Patronas, Nicholas J.; Schiffmann, Raphael; Brooks, Brian P.; Tamura, Deborah; DiGiovanna, John J.

2008-01-01

Patients with the rare genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) have defects in DNA nucleotide excision repair (NER). The NER pathway involves at least 28 genes. Three NER genes are also part of the basal transcription factor, TFIIH. Mutations in 11 NER genes have been associated with clinical diseases with at least 8 overlapping phenotypes. The clinical features of these patients have some similarities and but also have marked differences. NER is involved in protection against sunlight induced DNA damage. While XP patients have 1000-fold increase in susceptibility to skin cancer, TTD and CS patients have normal skin cancer risk. Several of the genes involved in NER also affect somatic growth and development. Some patients have short stature and immature sexual development. TTD patients have sulfur deficient brittle hair. Progressive sensorineural deafness is an early feature of XP and CS. Many of these clinical diseases are associated with developmental delay and progressive neurological degeneration. The main neuropathology of XP is a primary neuronal degeneration. In contrast, CS and TTD patients have reduced myelination of the brain. These complex neurological abnormalities are not related to sunlight exposure but may be caused by developmental defects as well as faulty repair of DNA damage to neuronal cells induced by oxidative metabolism or other endogenous processes. PMID:17276014

Mec1/ATR, the Program Manager of Nucleic Acids Inc.

PubMed

Feng, Wenyi

2016-12-28

Eukaryotic cells are equipped with surveillance mechanisms called checkpoints to ensure proper execution of cell cycle events. Among these are the checkpoints that detect DNA damage or replication perturbations and coordinate cellular activities to maintain genome stability. At the forefront of damage sensing is an evolutionarily conserved molecule, known respectively in budding yeast and humans as Mec1 (Mitosis entry checkpoint 1) and ATR (Ataxia telangiectasia and Rad3-related protein). Through phosphorylation, Mec1/ATR activates downstream components of a signaling cascade to maintain nucleotide pool balance, protect replication fork integrity, regulate activation of origins of replication, coordinate DNA repair, and implement cell cycle delay. This list of functions continues to expand as studies have revealed that Mec1/ATR modularly interacts with various protein molecules in response to different cellular cues. Among these newly assigned functions is the regulation of RNA metabolism during checkpoint activation and the coordination of replication-transcription conflicts. In this review, I will highlight some of these new functions of Mec1/ATR with a focus on the yeast model organism.

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.

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.

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis

PubMed Central

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura

2015-01-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. PMID:25986610

The autotaxin-LPA2 GPCR axis is modulated by γ-irradiation and facilitates DNA damage repair.

PubMed

Balogh, Andrea; Shimizu, Yoshibumi; Lee, Sue Chin; Norman, Derek D; Gangwar, Ruchika; Bavaria, Mitul; Moon, ChangSuk; Shukla, Pradeep; Rao, Radakrishna; Ray, Ramesh; Naren, Anjaparavanda P; Banerjee, Souvik; Banerje, Souvik; Miller, Duane D; Balazs, Louisa; Pelus, Louis; Tigyi, Gabor

2015-09-01

In this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)-LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin(-)Sca-1(+)c-Kit(+) enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair. Copyright © 2015. Published by Elsevier Inc.

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.

Effect of caffeine on the expression of a major X-ray induced protein in human tumor cells

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

Hughes, E.N.; Boothman, D.A.

1991-03-01

We have examined the effect of caffeine on the concomitant processes of the repair of potentially lethal damage (PLD) and the synthesis of X-ray-induced proteins in the human malignant melanoma cell line, Ul-Mel. Caffeine administered at a dose of 5mM after X radiation not only inhibited PLD repair but also markedly reduced the level of XIP269, a major X-ray-induced protein whose expression has been shown to correlate with the capacity to repair PLD. The expression of the vast majority of other cellular proteins, including seven other X-ray-induced proteins, remained unchanged following caffeine treatment. A possible role for XIP269 in cellmore » cycle delay following DNA damage by X irradiation is discussed.« less

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.

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.

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

Accumulation and developmental toxicity of hexabromocyclododecanes (HBCDs) on the marine copepod Tigriopus japonicus.

PubMed

Shi, Dalin; Lv, Dongmei; Liu, Wanxin; Shen, Rong; Li, Dongmei; Hong, Haizheng

2017-01-01

The brominated flame retardants hexabromocyclododecanes (HBCDs) are ubiquitous environmental contaminants, widely distributed in aquatic systems including the marine environment and marine organisms. HBCDs are toxic to the development of both freshwater and marine fish. However, the impacts of HBCDs on marine invertebrates are not well known. In this study, the marine copepod, Tigriopus japonicus, was used to assess the bioaccumulation and developmental toxicity of technical HBCD (tHBCD) through water-borne exposure. The uptake rate constant of tHBCD by T. japonicus was high, which resulted in high bioaccumulation potential. The bioconcentration factors of tHBCD were 8.73 × 10 4 and 6.34 × 10 4  L kg -1 in T. japonicus, calculated using the kinetic and steady-state methods, respectively. Exposure of T. japonicus nauplii to tHBCD caused significant growth delay. The lowest-observable-effect-concentrations of tHBCD induced developmental delay were 30 and 8 μg L -1 for the F0 and F1 generations, respectively, which suggested that the F1 generation was more sensitive to tHBCD than the F0 generation and warranted multiple-generation toxicity tests for future studies. Furthermore, exposure of the adult copepods to tHBCD induced the transcription of oxidative stress response genes and apoptotic genes, e.g., SOD,CAT, GST, OGG1, P53 and Caspase-3. It was therefore speculated that tHBCD exposure induced the generation of reactive oxygen species in T. japonicus, which activated the oxidative stress defense genes and meanwhile resulted in oxidative DNA damage. The damaged DNA activated the transcription of p53 and triggered the caspase-mediated apoptosis pathway, which may be the reason for the tHBCD induced developmental delay in T. japonicus nauplii. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

PubMed

Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

2015-08-01

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. 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.

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

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.

Inhibition of Ced-3/ICE-related Proteases Does Not Prevent Cell Death Induced by Oncogenes, DNA Damage, or the Bcl-2 Homologue Bak

PubMed Central

McCarthy, Nicola J.; Whyte, Moira K.B.; Gilbert, Christopher S.; Evan, Gerard I.

1997-01-01

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1β– converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode “death” gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage–induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing. PMID:9008715

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

PubMed

Tsuda, Masataka; Cho, Kosai; Ooka, Masato; Shimizu, Naoto; Watanabe, Reiko; Yasui, Akira; Nakazawa, Yuka; Ogi, Tomoo; Harada, Hiroshi; Agama, Keli; Nakamura, Jun; Asada, Ryuta; Fujiike, Haruna; Sakuma, Tetsushi; Yamamoto, Takashi; Murai, Junko; Hiraoka, Masahiro; Koike, Kaoru; Pommier, Yves; Takeda, Shunichi; Hirota, Kouji

2017-01-01

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

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.

Bioeffects of low-energy continuous ultrasound on isolated sarcoma 180 cells.

PubMed

Wang, Xiaobing; Liu, Quanhong; Wang, Zhezhi; Wang, Pan; Hao, Qiao; Li, Chendi

2009-01-01

The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis. Copyright 2009 S. Karger AG, Basel.

Topoisomerase IIα maintains genomic stability through decatenation G2 checkpoint signaling

PubMed Central

Bower, Jacquelyn J.; Karaca, Gamze F.; Zhou, Yingchun; Simpson, Dennis A.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

Topoisomerase IIα (topoIIα) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G2 checkpoint response, which delays mitotic entry due to insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIα and topoIIβ on decatenatory activity in cell extracts, DNA damage and decatenation G2 checkpoint function, and the frequencies of p16INK4A allele loss and gain. In diploid human fibroblast lines, depletion of topoIIα by siRNA was associated with severely reduced decatenatory activity, delayed progression from G2 into mitosis, and insensitivity to G2 arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIα-depleted cells displayed increased frequencies of losses and gains of the tumor suppressor genetic locus p16INK4A. This study demonstrates that the topoIIα protein is required for decatenation G2 checkpoint function, and inactivation of decatenation and the decatenation G2 checkpoint leads to abnormal chromosome segregation and genomic instability. PMID:20562910

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

PubMed

Pai, Chen-Chun; Kishkevich, Anastasiya; Deegan, Rachel S; Keszthelyi, Andrea; Folkes, Lisa; Kearsey, Stephen E; De León, Nagore; Soriano, Ignacio; de Bruin, Robertus Antonius Maria; Carr, Antony M; Humphrey, Timothy C

2017-09-12

Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

Suppression of Non-Homologous End Joining Repair by Overexpression of HMGA2

PubMed Central

Li, Angela Y.J.; Boo, Lee Ming; Wang, Shih-Ya; Lin, H. Helen; Wang, Clay C.C.; Yen, Yun; Chen, Benjamin P.C.; Chen, David J.; Ann, David K.

2009-01-01

Understanding the molecular details associated with aberrant high mobility group A2 (HMGA2) gene expression is key to establishing the mechanism(s) underlying its oncogenic potential and impact on the development of therapeutic strategies. Here, we report the involvement of HMGA2 in impairing DNA-dependent protein kinase (DNA-PK) during the non-homologous end joining (NHEJ) process. We demonstrated that HMGA2-expressing cells displayed deficiency in overall and precise DNA end-joining repair and accumulated more endogenous DNA damage. Proper and timely activation of DNA-PK, consisting of Ku70, Ku80 and DNA-PKcs subunits, is essential for the repair of DNA double strand breaks (DSBs) generated endogenously or by exposure to genotoxins. In cells overexpressing HMGA2, accumulation of histone 2A variant X phosphorylation at Ser-139 (γ-H2AX) was associated with hyper-phosphorylation of DNA-PKcs at Thr-2609 and Ser-2056 before and after the induction of DSBs. Also, the steady-state complex of Ku and DNA ends was altered by HMGA2. Microirradiation and real-time imaging in living cells revealed that HMGA2 delayed the release of DNA-PKcs from DSB sites, similar to observations found in DNA-PKcs mutants. Moreover, HMGA2 alone was sufficient to induce chromosomal aberrations, a hallmark of deficiency in NHEJ-mediated DNA repair. In summary, a novel role for HMGA2 to interfere with NHEJ processes was uncovered, implicating HMGA2 in the promotion of genome instability and tumorigenesis. PMID:19549901

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

Contribution of sleep to the repair of neuronal DNA double-strand breaks: evidence from flies and mice.

PubMed

Bellesi, Michele; Bushey, Daniel; Chini, Mattia; Tononi, Giulio; Cirelli, Chiara

2016-11-10

Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether sleep provides any selective advantage over wake in their repair. In flies and mice, we find that enriched wake, more than simply time spent awake, induces DSBs, and their repair in mice is delayed or prevented by subsequent wake. In both species the repair of irradiation-induced neuronal DSBs is also quicker during sleep, and mouse genes mediating the response to DNA damage are upregulated in sleep. Thus, sleep facilitates the repair of neuronal DSBs.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair

PubMed Central

Morales, Julio C.; Richard, Patricia; Rommel, Amy; Fattah, Farjana J.; Motea, Edward A.; Patidar, Praveen L.; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N.; Chiang, Cheng-Ming; Manley, James L.; Boothman, David A.

2014-01-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair. PMID:24589584

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

PubMed

Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

2014-04-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

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.

Assessment of the role of DNA repair in damaged forensic samples.

PubMed

Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; King, Jonathan; Budowle, Bruce

2014-11-01

Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results.

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

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.

DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

NASA Astrophysics Data System (ADS)

Han, Xu; Klas, Matej; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

2013-09-01

The nitrogen atmospheric pressure plasma jet (APPJ) has been shown to effectively induce DNA double strand breaks in SCC-25 oral cancer cells. The APPJ source constructed in our laboratory consists of two external electrodes wrapping around a quartz tube and nitrogen as a feed gas and operates based on dielectric barrier gas discharge. Generally, it is more challenging to ignite plasma in N2 atmosphere than in noble gases. However, this design provides additional advantages such as lower costs compared to the noble gases for future clinical operation. Different parameters of the APPJ configuration were tested in order to determine radiation dosage. To explore the effects of delayed damage and cell self-repairing, various incubation times of cells after plasma treatment were also performed. Reactive species generated in plasma jet and in liquid environment are essential to be identified and quantified, with the aim of unfolding the mystery of detailed mechanisms for plasma-induced cell apoptosis. Moreover, from the comparison of plasma treatment effect on normal oral cells OKF6T, an insight to the selectivity for cancer treatment by APPJ can be explored. All of these studies are critical to better understand the damage responses of normal and abnormal cellular systems to plasma radiation, which are useful for the development of advanced plasma therapy for cancer treatment at a later stage.

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

PubMed

Kamenšek, Simona; Browning, Douglas F; Podlesek, Zdravko; Busby, Stephen J W; Žgur-Bertok, Darja; Butala, Matej

2015-06-01

Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

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

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

PubMed

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

2015-08-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Gene expression profiling in human skeletal muscle during recovery from eccentric exercise

PubMed Central

Mohoney, D. J.; Safdar, A.; Parise, G.; Melov, S.; Fu, Minghua; MacNeil, L.; Kaczor, J.; Payne, E. T.; Tarnopolsky, M. A.

2009-01-01

We used cDNA microarrays to screen for differentially expressed genes during recovery from exercise-induced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using real-time RT-PCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Real-time RT-PCR analyses confirmed that exercise-induced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP-2), followed by a delayed (48 h) increase in the SREBP-2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)-2 and insulin-induced gene 1 (insig-1). The expression of the IL-1 receptor, a known regulator of SREBP-2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZα), and modulatory calcineurin-interacting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, c-myc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RT-PCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise. PMID:18321953

Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure

PubMed Central

Guillonneau, F.; Guieysse, A. L.; Nocentini, S.; Giovannangeli, C.; Praseuth, D.

2004-01-01

Targeting DNA-damaging agents to specific DNA sites by using sequence-specific DNA ligands has been successful in directing genomic modifications. The understanding of repair processing of such targeted damage and the influence of the adjacent complex is largely unknown. In this way, directed interstrand cross-links (ICLs) have already been generated by psoralen targeting. The mechanisms responsible for ICL removal are far from being understood in mammalian cells, with the proposed involvement of both mutagenic and recombinogenic pathways. Here, a unique ICL was introduced at a selected site by photoactivation of a psoralen moiety with the use of psoralen conjugates of triplex-forming oligonucleotides. The processing of psoralen ICL was evaluated in vitro and in cells for two types of cross-linked substrates, either containing a psoralen ICL alone or with an adjacent triple-stranded structure. We show that the presence of a neighbouring triplex structure interferes with different stages of psoralen ICL processing: (i) the ICL-induced DNA repair synthesis in HeLa cell extracts is inhibited by the triplex structure, as measured by the efficiency of ‘true’ and futile repair synthesis, stopping at the ICL site; (ii) in HeLa cells, the ICL removal via a nucleotide excision repair (NER) pathway is delayed in the presence of a neighbouring triplex; and (iii) the binding to ICL of recombinant xeroderma pigmentosum A protein, which is involved in pre-incision recruitment of NER factors is impaired by the presence of the third DNA strand. These data characterize triplex-induced modulation of ICL repair pathways at specific steps, which might have implications for the controlled induction of targeted genomic modifications and for the associated cellular responses. PMID:14966263

Mutations in SID2, a novel gene in Saccharomyces cerevisiae, cause synthetic lethality with sic1 deletion and may cause a defect during S phase.

PubMed Central

Jacobson, M D; Muñoz, C X; Knox, K S; Williams, B E; Lu, L L; Cross, F R; Vallen, E A

2001-01-01

SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis. To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1 Delta were isolated. In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair. sid2-1 sic1 Delta strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an approximately 2C DNA content. RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1 Delta cells. Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest. Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1 Delta inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1 Delta cells. In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed. sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability. Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed. PMID:11560884

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

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

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.

Two regulatory RNA elements affect TisB-dependent depolarization and persister formation.

PubMed

Berghoff, Bork A; Hoekzema, Mirthe; Aulbach, Lena; Wagner, E Gerhart H

2017-03-01

Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5' UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity. © 2016 John Wiley & Sons Ltd.

Feedback between p21 and reactive oxygen production is necessary for cell senescence

PubMed Central

Passos, João F; Nelson, Glyn; Wang, Chunfang; Richter, Torsten; Simillion, Cedric; Proctor, Carole J; Miwa, Satomi; Olijslagers, Sharon; Hallinan, Jennifer; Wipat, Anil; Saretzki, Gabriele; Rudolph, Karl Lenhard; Kirkwood, Tom B L; von Zglinicki, Thomas

2010-01-01

Cellular senescence—the permanent arrest of cycling in normally proliferating cells such as fibroblasts—contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of ‘deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFβ. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype. PMID:20160708

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

Increased Arf/p53 activity in stem cells, aging and cancer.

PubMed

Carrasco-Garcia, Estefania; Moreno, Manuel; Moreno-Cugnon, Leire; Matheu, Ander

2017-04-01

Arf/p53 pathway protects the cells against DNA damage induced by acute stress. This characteristic is the responsible for its tumor suppressor activity. Moreover, it regulates the chronic type of stress associated with aging. This is the basis of its anti-aging activity. Indeed, increased gene dosage of Arf/p53 displays elongated longevity and delayed aging. At a cellular level, it has been recently shown that increased dosage of Arf/p53 delays age-associated stem cell exhaustion and the subsequent decline in tissue homeostasis and regeneration. However, p53 can also promote aging if constitutively activated. In this context, p53 reduces tissue regeneration, which correlates with premature exhaustion of stem cells. We discuss here the current evidence linking the Arf/p53 pathway to the processes of aging and cancer through stem cell regulation. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Insulin and insulin-like growth factor-1 (lGF-1) inhibit repair of potentially lethal radiation damage and chromosome aberrations and later DNA repair kinetics in plateau-phase A549 cells

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

Jayanth, V.R.; Belfi, C.A.; Swick, A.R.

1995-08-01

Plateau-phase A549 cells exhibit a high capacity for repair of potentially lethal radiation damage (PLD) when allowed to recover in their own spent medium. Addition of either insulin or insulin-like growth factor-1 (IGF-1) to the spent medium 60 to 120 min before irradiation significantly inhibits PLD repair. The 9-h recovery factor (survival with holding/survival without holding)is reduced from 10.8 {plus_minus} 0.7 to 3.4 {plus_minus}0.3 by insulin and to 3.0 {plus_minus} 0.4 by IGF-1. Neither growth factor alters the cell age distribution of the plateau-phase cells, increases the rate of incorporation of 5-bromo-2{prime}-deoxyuridine into DNA, or alters the extent of radiation-inducedmore » mitotic delay in cells subcultured immediately after irradiation. Both insulin and IGF-1 alter the kinetics for rejoining of DNA double-strand breaks (DSBs), slowing the fast component of rejoining significantly. However, these growth factors have no effect on the initial level of DSBs or on the percentage of residual unrejoined breaks at 120 min postirradiation. Both growth factors affect repair of lesions leading to dicentric, but not to acentric, chromosome aberrations significantly. In control cells (treated with phosphate-buffered saline, 90 min prior to irradiation), the half-time for disappearance of dicentrics was 4.1 h (3.4 to 5.1 h), and 47.1 {plus_minus} 3.7% of the residual damage remained at 24 h postirradiation. Insulin and IGF-1 increased the half-time for disappearance of dicentrics to 5.2 h (3.9 to 7.7 h) and 5.7 h (5.5 to 5.9 h), respectively, and increased residual damage to 56.1 {plus_minus}5.9% and 60.8 {plus_minus} 6.0%, respectively. Overall, these data show that insulin and IGF-1 inhibit PLD repair in A54j9 cells by mechanisms which are independent of changes in cell cycle parameters. The data suggest that the growth factors act by inducing changes in chromatin conformation which promote misrepair of radiation-damaged DNA. 49 refs., 5 figs., 4 tabs.« less

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

Initial characterization of a low-molecular-weight factor enhancing the checkpoint response.

PubMed

Fan, Xiaoxiang; Cheong, Nge; Iliakis, George

2010-10-01

In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity. Although compounds are known that reduce the G(2) checkpoint and act as radiosensitizers, compounds enhancing this checkpoint have not been reported. Here we summarize evidence for a factor with such properties. We show that a highly radioresistant rat embryo fibroblast (REF) cell line displays a strong G(2) checkpoint partly as a result of a factor excreted into the growth medium by nonirradiated cells. Various tests indicate that this G(2)-arrest modulating activity (GAMA) is a small molecule showing detectable retention only after passing through filters with a molecular weight cutoff limit of less than 1,000 Da. GAMA is heat stable and resistant to treatment with proteases or nucleases. Electroelution tests show that GAMA is uncharged at neutral pH, a result that is in agreement with the observed failure to bind S- or Q-Sepharose. Investigations on the mechanism of GAMA function indicate ligand-receptor interactions and allow the classification of cells as producers, responders or both. Compounds with properties such as those of GAMA bridge intercellular communication with the DNA damage response and may function as radioprotectors.

Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom's syndrome gene.

PubMed

Chester, N; Kuo, F; Kozak, C; O'Hara, C D; Leder, P

1998-11-01

Bloom's syndrome is a human autosomal genetic disorder characterized at the cellular level by genome instability and increased sister chomatid exchanges (SCEs). Clinical features of the disease include proportional dwarfism and a predisposition to develop a wide variety of malignancies. The human BLM gene has been cloned recently and encodes a DNA helicase. Mouse embryos homozygous for a targeted mutation in the murine Bloom's syndrome gene (Blm) are developmentally delayed and die by embryonic day 13.5. The fact that the interrupted gene is the homolog of the human BLM gene was confirmed by its homologous sequence, its chromosomal location, and by demonstrating high numbers of SCEs in cultured murine Blm-/- fibroblasts. The proportional dwarfism seen in the human is consistent with the small size and developmental delay (12-24 hr) seen during mid-gestation in murine Blm-/- embryos. Interestingly, the growth retardation in mutant embryos can be accounted for by a wave of increased apoptosis in the epiblast restricted to early post-implantation embryogenesis. Mutant embryos do not survive past day 13.5, and at this time exhibit severe anemia. Red blood cells and their precursors from Blm-/- embryos are heterogeneous in appearance and have increased numbers of macrocytes and micronuclei. Both the apoptotic wave and the appearance of micronuclei in red blood cells are likely cellular consequences of damaged DNA caused by effects on replicating or segregating chromosomes.

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.

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.

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.

CuInS2/ZnS QD exposure induces developmental toxicity, oxidative stress and DNA damage in rare minnow (Gobiocypris rarus) embryos and larvae.

PubMed

Liu, Li; Xiao, Yuan-Yuan; Ji, Yan-Hong; Liu, Ming-Zhi; Chen, Yao; Zeng, Yu-Lian; Zhang, Yao-Guang; Jin, Li

2017-08-01

Chinese rare minnow (Gobiocypris rarus) embryos were used as an experimental model to investigate the effects of CuInS 2 /ZnS quantum dots (QDs) on the early life stages of G. rarus. Normal developmental parameters (survival rate, body length and average heart rate), biomarker genes [stress response (Hsp70), detoxification (Cyp1a), organizer function and axis formation (Wnt8α), and muscle (Mstn)], enzymatic activity and DNA damage were recorded as endpoints in the developing embryos/larvae after exposure until 96h post-fertilization (hpf). Reduced survival rate, decreased heart rate, altered body length, increased malformation rate, decreased hatching rate, advanced hatching time in response to low concentrations (50 and 100nmol/L) and delayed hatching time in response to high concentrations were observed after exposure, as were many other toxic effects, including pericardial edema and bent tails. The 72 hpf LC 50 (median lethal concentration) was determined to be 624.364nmol/L. Treatment with certain concentrations of CuInS 2 /ZnS QDs significantly increased the superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels and significantly induced DNA damage. After treatment with CuInS 2 /ZnS QDs, the embryos showed highly up-regulated expression of Hsp70, Cyp1a and Wnt8a and significantly up-regulated expression of Mstn at 12 hpf. Overall, this study indicates that CuInS 2 /ZnS QDs are potentially toxic to G. rarus embryos. The information presented in this study will be helpful for fully understanding the toxicity induced by CuInS 2 /ZnS QDs in fish embryos. Copyright © 2017 Elsevier Inc. All rights reserved.

Adamantanyl-Histone Deacetylase Inhibitor H6CAHA Exhibits Favorable Pharmacokinetics and Augments Prostate Cancer Radiation Sensitivity

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

Konsoula, Zacharoula; Cao Hong; Velena, Alfredo

2011-04-01

Purpose: To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to {gamma}-radiation in vitro and in vivo. Methods and Materials: H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice. Results: H6CAHA demonstrated goodmore » solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 {+-} 0.91 {mu}M x h, and its half-life was 11.17 {+-} 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of {gamma}H2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with {gamma}-radiation (10 x 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days. Conclusion: These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation.« less

A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells.

PubMed

Wei, Ren-Jie; Lin, Su-Shuan; Wu, Wen-Ren; Chen, Lih-Ren; Li, Chien-Feng; Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun; Liang, Shih-Shin; Chien, Shang-Tao; Shiue, Yow-Ling

2016-11-15

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G 2 /M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G 2 /M cell cycle regulators, ABT-751 induced G 2 /M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. Copyright © 2016 Elsevier Inc. All rights reserved.

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 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

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

PubMed Central

Hrebien, Sarah; O’Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Turner, Nicholas

2016-01-01

Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research. PMID:27760227

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer.

PubMed

Hrebien, Sarah; O'Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Garcia-Murillas, Isaac; Turner, Nicholas

2016-01-01

Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48-72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77-0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research.

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.

Vitamin E Supplementation Reduces Cellular Loss in the Brain of a Premature Aging Mouse Model.

PubMed

La Fata, G; van Vliet, N; Barnhoorn, S; Brandt, R M C; Etheve, S; Chenal, E; Grunenwald, C; Seifert, N; Weber, P; Hoeijmakers, J H J; Mohajeri, M H; Vermeij, W P

2017-01-01

Aging is a highly complex biological process driven by multiple factors. Its progression can partially be influenced by nutritional interventions. Vitamin E is a lipid-soluble anti-oxidant that is investigated as nutritional supplement for its ability to prevent or delay the onset of specific aging pathologies, including neurodegenerative disorders. We aimed here to investigate the effect of vitamin E during aging progression in a well characterized mouse model for premature aging. Xpg-/- animals received diets with low (~2.5 mg/kg feed), medium (75 mg/kg feed) or high (375 mg/kg feed) vitamin E concentration and their phenotype was monitored during aging progression. Vitamin E content was analyzed in the feed, for stability reasons, and in mouse plasma, brain, and liver, for effectiveness of the treatment. Subsequent age-related changes were monitored for improvement by increased vitamin E or worsening by depletion in both liver and nervous system, organs sensitive to oxidative stress. Mice supplemented with high levels of vitamin E showed a delayed onset of age-related body weight decline and appearance of tremors when compared to mice with a low dietary vitamin E intake. DNA damage resulting in liver abnormalities such as changes in polyploidy, was considerably prevented by elevated amounts of vitamin E. Additionally, immunohistochemical analyses revealed that high intake of vitamin E, when compared with low and medium levels of vitamin E in the diet, reduces the number of p53-positive cells throughout the brain, indicative of a lower number of cells dying due to DNA damage accumulated over time. Our data underline a neuroprotective role of vitamin E in the premature aging animal model used in this study, likely via a reduction of oxidative stress, and implies the importance of improved nutrition to sustain health.

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.

Indirect macrophage responses to ionizing radiation: implications for genotype-dependent bystander signaling.

PubMed

Coates, Philip J; Rundle, Jana K; Lorimore, Sally A; Wright, Eric G

2008-01-15

In addition to the directly mutagenic effects of energy deposition in DNA, ionizing radiation is associated with a variety of untargeted and delayed effects that result in ongoing bone marrow damage. Delayed effects are genotype dependent with CBA/Ca mice, but not C57BL/6 mice, susceptible to the induction of damage and also radiation-induced acute myeloid leukemia. Because macrophages are a potential source of ongoing damaging signals, we have determined their gene expression profiles and we show that bone marrow-derived macrophages show widely different intrinsic expression patterns. The profiles classify macrophages derived from CBA/Ca mice as M1-like (pro-inflammatory) and those from C57BL/6 mice as M2-like (anti-inflammatory); measurements of NOS2 and arginase activity in normal bone marrow macrophages confirm these findings. After irradiation in vivo, but not in vitro, C57BL/6 macrophages show a reduction in NOS2 and an increase in arginase activities, indicating a further M2 response, whereas CBA/Ca macrophages retain an M1 phenotype. Activation of specific signal transducer and activator of transcription signaling pathways in irradiated hemopoietic tissues supports these observations. The data indicate that macrophage activation is not a direct effect of radiation but a tissue response, secondary to the initial radiation exposure, and have important implications for understanding genotype-dependent responses and the mechanisms of the hemotoxic and leukemogenic consequences of radiation exposure.

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...

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.

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.

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

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

PubMed

Toledo, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Claudia; Larsen, Dorthe Helena; Povlsen, Lou Klitgaard; Bekker-Jensen, Simon; Mailand, Niels; Bartek, Jiri; Lukas, Jiri

2013-11-21

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

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.

Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro

PubMed Central

Purohit, Nupur K.; Robu, Mihaela; Shah, Rashmi G.; Geacintov, Nicholas E.; Shah, Girish M.

2016-01-01

The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA. PMID:26753915

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

Chemical determination of free radical-induced damage to DNA.

PubMed

Dizdaroglu, M

1991-01-01

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

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.

N-acetylcysteine protects melanocytes against oxidative stress/damage and delays onset of UV-induced melanoma in mice

PubMed Central

Cotter, Murray A.; Thomas, Joshua; Cassidy, Pamela; Robinette, Kyle; Jenkins, Noah; Scott, R. Florell; Leachman, Sancy; Samlowski, Wolfram E.; Grossman, Douglas

2008-01-01

UV radiation is the major environmental risk factor for melanoma and a potent inducer of oxidative stress, which is implicated in the pathogenesis of several malignancies. We evaluated whether the thiol antioxidant N-acetylcysteine (NAC) could protect melanocytes from UV-induced oxidative stress/damage in vitro and from UV-induced melanoma in vivo. In melan-a cells, a mouse melanocyte line, NAC (1–10 mM) conferred protection from several UV-induced oxidative sequelae including production of intracellular peroxide, formation of the signature oxidative DNA lesion 8-oxoguanine (8-OG), and depletion of free reduced thiols (primarily glutathione). Mice transgenic for hepatocyte growth factor and Survivin, previously shown to develop melanoma following a single neonatal dose of UV irradiation, were administered NAC (7 mg/ml, mother’s drinking water) transplacentally and through nursing until two weeks after birth. Delivery of NAC in this manner reduced thiol depletion and blocked formation of 8-OG in skin following neonatal UV treatment. Mean onset of UV-induced melanocytic tumors was significantly delayed in NAC-treated compared to control mice (21 vs. 14 weeks, p=0.0003). Our data highlight the potential importance of oxidative stress in the pathogenesis of melanoma, and suggest that NAC may be useful as a chemopreventive agent. PMID:17908992

Cisplatin resistance induced in germ cell tumour cells is due to reduced susceptibility towards cell death but not to altered DNA damage induction or repair.

PubMed

Fenske, Annabelle E; Glaesener, Stephanie; Bokemeyer, Carsten; Thomale, Juergen; Dahm-Daphi, Jochen; Honecker, Friedemann; Dartsch, Dorothee C

2012-11-28

To identify factors involved in cisplatin (CDDP) resistance of germ cell tumours (GCTs), we exposed NTERA-2 cells, and the platinum-adapted subline NTERA-2R to CDDP and compared their response. While both cell lines showed comparable proliferation, NTERA-2R cells were clearly more resistant to the drug than the parental NTERA-2 cell line. Interestingly, the two lines showed identical extent of DNA adduct formation and elimination, indicating that neither changes in CDDP uptake, nor altered drug efflux, DNA binding, or repair caused the difference in resistance. Similarly, no difference occurred in the time-course of γH2AX formation, which was not linked to 53BP1 accumulation. In contrast, NTERA-2R cells showed a more pronounced dose-dependent S phase delay, a transient G(2)/M-block, and subsequent release into immediate cell death. We thus conclude that the enhanced resistance against CDDP is linked to reduced susceptibility to cell death rather than to an altered DNA adduct formation or adduct removal. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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.

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.

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

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.

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

How Human Papillomavirus Replication and Immune Evasion Strategies Take Advantage of the Host DNA Damage Repair Machinery

PubMed Central

Bordignon, Valentina; Trento, Elisabetta; D’Agosto, Giovanna; Cavallo, Ilaria; Pontone, Martina; Pimpinelli, Fulvia; Mariani, Luciano; Ensoli, Fabrizio

2017-01-01

The DNA damage response (DDR) is a complex signalling network activated when DNA is altered by intrinsic or extrinsic agents. DDR plays important roles in genome stability and cell cycle regulation, as well as in tumour transformation. Viruses have evolved successful life cycle strategies in order to ensure a chronic persistence in the host, virtually avoiding systemic sequelae and death. This process promotes the periodic shedding of large amounts of infectious particles to maintain a virus reservoir in individual hosts, while allowing virus spreading within the community. To achieve such a successful lifestyle, the human papilloma virus (HPV) needs to escape the host defence systems. The key to understanding how this is achieved is in the virus replication process that provides by itself an evasion mechanism by inhibiting and delaying the host immune response against the viral infection. Numerous studies have demonstrated that HPV exploits both the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) DDR pathways to replicate its genome and maintain a persistent infection by downregulating the innate and cell-mediated immunity. This review outlines how HPV interacts with the ATM- and ATR-dependent DDR machinery during the viral life cycle to create an environment favourable to viral replication, and how the interaction with the signal transducers and activators of transcription (STAT) protein family and the deregulation of the Janus kinase (JAK)–STAT pathways may impact the expression of interferon-inducible genes and the innate immune responses. PMID:29257060

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.

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.

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

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

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.

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

X-RAY INDUCED INCORPORATION OF TRITIATED THYMIDINE INTO GRASSHOPPER NEUROBLAST CHROMOSOMES

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

McGrath, R.A.

1962-01-01

Neuroblasts of the grasshopper Chortophage viridifasciata (De Geer) were used to study incorporation of tritiated thymidine (H/sub 3/Thd), a deoxyribonucleic acid (DNA) precursor, into chromosomes. Embryos were made into hanging drop cultures which contained H/sub 3/Thd and exposed to 32 or 250 r of x rays. Individual cells were identified and observed by bright field microscopy for periods of time which ranged from 15 min to 6 hrs after irradiation. At the end of the observation period embryos were either sectioned or made into squash preparations. In the former case preselected neuroblasts were reidentified: in the latter they were not.more » Results of observations of living neuroblasts in hanging drop cultures are presented. Autoradiograms were prepared for the assay of H/sub 3/Thd incorporation by grain counting methods. Information obtained from the autoradiograms is discussed. Results are interpreted as a reflection of repair of x-ray-damaged DNA. A correlation is suggested between detectable chromosome breakage, the normal DNA synthesis period, and labeling of delayed, stopped or reverted prophase neuroblasts. (M.P.G.)« less

Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control.

PubMed

Tessadori, Federico; Giltay, Jacques C; Hurst, Jane A; Massink, Maarten P; Duran, Karen; Vos, Harmjan R; van Es, Robert M; Scott, Richard H; van Gassen, Koen L I; Bakkers, Jeroen; van Haaften, Gijs

2017-11-01

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

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.

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

Action of caffeine on x-irradiated HeLa cells. IV. Progression delays and enhanced cell killing at high caffeine concentrations

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

Tolmach, L.J.; Busse, P.M.

1980-05-01

The response of x-irradiated and unirradiated HeLa S3 cells to treatment with caffeine at concentrations between 1 and 10 nM has been examined with respect to both delay in progression through the cell generation cycle and enhancement of the expression of potentially lethal x-ray damage. Progression is delayed in a concentration-dependent fashion: the generation time is doubled at about 4 mM. The duration of G/sub 1/ is lengthened, and the rate of DNA synthesis is reduced, although the kinetics are different in the two phases; the rate of DNA synthesis is usually unaffected at 1 or 2 mM, while theremore » is no concentration threshold for the slowing of progression through G/sub 1/. Progression through G/sub 2/ appears to be unaffected by concentrations up to at least 10 mM. Killing of irradiated cells in G/sub 2/ is somewhat greater after treatment with the higher caffeine concentrations than reported previously for 1 mM. Moreover, an additional mode of killing is observed in irradiated G/sub 1/ cells which had been found previously to be only slightly affected by 1 mM caffeine; they suffer extensive killing at concentrations above 5 mM. The time-survival curves for irradiated, caffeine-treated G/sub 1/ and G/sub 2/ cells have characteristically different shapes. The dose-survival curves for cells treated with the higher caffeine concentrations display steeper terminal slopes and narrower shoulders.« less

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.

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.

The usefulness of cytogenetic parameters, level of p53 protein and endogenous glutathione as intermediate end-points in raw betel-nut genotoxicity.

PubMed

Kumpawat, K; Chatterjee, A

2003-07-01

Betel-nut (BN) chewing related oral mucosal lesions are potential hazards to a large population worldwide. Genotoxicity of betel alkaloids, polyphenol and tannin fractions have been reported. It has been shown earlier that BN ingredients altered the level of endogenous glutathione (GSH) which could modulate the host susceptibility to the action of other chemical carcinogens. The north-east Indian variety of BN, locally known as 'kwai', is raw, wet and consumed unprocessed with betel-leaf and slaked lime and contains higher alkaloids, polyphenol and tannins as compared to the dried one. Therefore, the purpose of this study was to investigate the extent of DNA damage, pattern of cell kinetics, the level of p53-protein and endogenous GSH in kwai chewers in the tribal population of Meghalaya state in the northeastern region of India with an aim to see whether these end-points could serve as biomarkers of genetic damage of relevance for genotoxic/carcinogenic process. The present data show higher DNA damage, delay in cell kinetics, p53 expression and lower GSH-level in heavy chewers (HC) than nonchewers (NC). The influence of bleomycin (BLM) on chromatid break induction in G2-phase of peripheral blood lymphocytes in NC and HC has been analysed to determine individual susceptibility to carcinogenic assaults. HC showed higher induction of chromatid breaks than NC. Risk assessment in this study suggests an interaction between carcinogen exposure and mutagen sensitivity measures, risk estimates being higher in those individuals who both consume kwai and express sensitivity to free radical oxygen damage in vitro. From this study it seems that besides cytogenetical parameters, the level of endogenous GSH and the level of p53 protein could act as effective biomarkers for kwai chewers.

Folic acid deficiency increases delayed neuronal death, DNA damage, platelet endothelial cell adhesion molecule-1 immunoreactivity, and gliosis in the hippocampus after transient cerebral ischemia.

PubMed

Hwang, In Koo; Yoo, Ki-Yeon; Suh, Hong-Won; Kim, Young Sup; Kwon, Dae Young; Kwon, Young-Guen; Yoo, Jun-Hyun; Won, Moo-Ho

2008-07-01

Folic acid deficiency increases stroke risk. In the present study, we examined whether folic acid deficiency enhances neuronal damage and gliosis via oxidative stress in the gerbil hippocampus after transient forebrain ischemia. Animals were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to occlusion of both common carotid arteries for 5 min. Exposure to an FAD increased plasma homocysteine levels by five- to eightfold compared with those of animals fed with a control diet (CD). In CD-treated animals, most neurons were dead in the hippocampal CA1 region 4 days after ischemia/reperfusion, whereas, in FAD-treated animals, this occurred 3 days after ischemia/reperfusion. Immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was performed to examine DNA damage in CA1 neurons in both groups after ischemia, and it was found that 8-OHdG immunoreactivity in both FAD and CD groups peaked at 12 hr after reperfusion, although the immunoreactivity in the FAD group was much greater than that in the CD group. Platelet endothelial cell adhesion molecule-1 (PECAM-1; a final mediator of neutrophil transendothelial migration) immunoreactivity in both groups increased with time after ischemia/reperfusion: Its immunoreactivity in the FAD group was much higher than that in the CD group 3 days after ischemia/reperfusion. In addition, reactive gliosis in the ischemic CA1 region increased with time after ischemia in both groups, but astrocytosis and microgliosis in the FAD group were more severe than in the CD group at all times after ischemia. Our results suggest that folic acid deficiency enhances neuronal damage induced by ischemia. 2008 Wiley-Liss, Inc.

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

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.

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.

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.

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

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.

Free Radicals Generated by Ionizing Radiation Signal Nuclear Translocation of p53

NASA Technical Reports Server (NTRS)

Martinez, J. D.; Pennington, M. E.; Craven, M. T.; Warters, R. L.

1997-01-01

The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p63 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 hours postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 hours after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.

Prevention of Mutation, Cancer, and Other Age-Associated Diseases by Optimizing Micronutrient Intake

PubMed Central

Ames, Bruce N.

2010-01-01

I review three of our research efforts which suggest that optimizing micronutrient intake will in turn optimize metabolism, resulting in decreased DNA damage and less cancer as well as other degenerative diseases of aging. (1) Research on delay of the mitochondrial decay of aging, including release of mutagenic oxidants, by supplementing rats with lipoic acid and acetyl carnitine. (2) The triage theory, which posits that modest micronutrient deficiencies (common in much of the population) accelerate molecular aging, including DNA damage, mitochondrial decay, and supportive evidence for the theory, including an in-depth analysis of vitamin K that suggests the importance of achieving optimal micronutrient intake for longevity. (3) The finding that decreased enzyme binding constants (increased Km) for coenzymes (or substrates) can result from protein deformation and loss of function due to an age-related decline in membrane fluidity, or to polymorphisms or mutation. The loss of enzyme function can be compensated by a high dietary intake of any of the B vitamins, which increases the level of the vitamin-derived coenzyme. This dietary remediation illustrates the importance of understanding the effects of age and polymorphisms on optimal micronutrient requirements. Optimizing micronutrient intake could have a major effect on the prevention of cancer and other degenerative diseases of aging. PMID:20936173

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.

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.

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

Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols.

PubMed

Wang, Amy; Robertson, John L; Holladay, Steven D; Tennant, Alan H; Lengi, Andrea J; Ahmed, S Ansar; Huckle, William R; Kligerman, Andrew D

2007-12-01

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin-EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA-protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.

Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success.

PubMed

Pérez-Cerezales, S; Martínez-Páramo, S; Beirão, J; Herráez, M P

2010-06-01

Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported.

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.

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...

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

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.

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

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.

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

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.

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.

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

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

PubMed

Lee, Ji-Hoon; Mand, Michael R; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W; Richards, Alicia L; Coon, Joshua J; Paull, Tanya T

2018-01-09

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. We genetically separated the activation of ATM by DNA damage from that by oxidative stress using separation-of-function mutations. We found that deficient activation of ATM by the Mre11-Rad50-Nbs1 complex and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of a variant ATM incapable of activation by oxidative stress resulted in widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicate ATM in the control of protein homeostasis. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Ionizing radiation from Chernobyl affects development of wild carrot plants.

PubMed

Boratyński, Zbyszek; Arias, Javi Miranda; Garcia, Cristina; Mappes, Tapio; Mousseau, Timothy A; Møller, Anders P; Pajares, Antonio Jesús Muñoz; Piwczyński, Marcin; Tukalenko, Eugene

2016-12-16

Radioactivity released from disasters like Chernobyl and Fukushima is a global hazard and a threat to exposed biota. To minimize the deleterious effects of stressors organisms adopt various strategies. Plants, for example, may delay germination or stay dormant during stressful periods. However, an intense stress may halt germination or heavily affect various developmental stages and select for life history changes. Here, we test for the consequence of exposure to ionizing radiation on plant development. We conducted a common garden experiment in an uncontaminated greenhouse using 660 seeds originating from 33 wild carrots (Daucus carota) collected near the Chernobyl nuclear power plant. These maternal plants had been exposed to radiation levels that varied by three orders of magnitude. We found strong negative effects of elevated radiation on the timing and rates of seed germination. In addition, later stages of development and the timing of emergence of consecutive leaves were delayed by exposure to radiation. We hypothesize that low quality of resources stored in seeds, damaged DNA, or both, delayed development and halted germination of seeds from plants exposed to elevated levels of ionizing radiation. We propose that high levels of spatial heterogeneity in background radiation may hamper adaptive life history responses.

Ionizing radiation from Chernobyl affects development of wild carrot plants

NASA Astrophysics Data System (ADS)

Boratyński, Zbyszek; Arias, Javi Miranda; Garcia, Cristina; Mappes, Tapio; Mousseau, Timothy A.; Møller, Anders P.; Pajares, Antonio Jesús Muñoz; Piwczyński, Marcin; Tukalenko, Eugene

2016-12-01

Radioactivity released from disasters like Chernobyl and Fukushima is a global hazard and a threat to exposed biota. To minimize the deleterious effects of stressors organisms adopt various strategies. Plants, for example, may delay germination or stay dormant during stressful periods. However, an intense stress may halt germination or heavily affect various developmental stages and select for life history changes. Here, we test for the consequence of exposure to ionizing radiation on plant development. We conducted a common garden experiment in an uncontaminated greenhouse using 660 seeds originating from 33 wild carrots (Daucus carota) collected near the Chernobyl nuclear power plant. These maternal plants had been exposed to radiation levels that varied by three orders of magnitude. We found strong negative effects of elevated radiation on the timing and rates of seed germination. In addition, later stages of development and the timing of emergence of consecutive leaves were delayed by exposure to radiation. We hypothesize that low quality of resources stored in seeds, damaged DNA, or both, delayed development and halted germination of seeds from plants exposed to elevated levels of ionizing radiation. We propose that high levels of spatial heterogeneity in background radiation may hamper adaptive life history responses.

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.

Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

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 refer to as 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. PMID:20469926

Response to DNA damage of CHEK2 missense mutations in familial breast cancer

PubMed Central

Roeb, Wendy; Higgins, Jake; King, Mary-Claire

2012-01-01

Comprehensive sequencing of tumor suppressor genes to evaluate inherited predisposition to cancer yields many individually rare missense alleles of unknown functional and clinical consequence. To address this problem for CHEK2 missense alleles, we developed a yeast-based assay to assess in vivo CHEK2-mediated response to DNA damage. Of 25 germline CHEK2 missense alleles detected in familial breast cancer patients, 12 alleles had complete loss of DNA damage response, 8 had partial loss and 5 exhibited a DNA damage response equivalent to that mediated by wild-type CHEK2. Variants exhibiting reduced response to DNA damage were found in all domains of the CHEK2 protein. Assay results were in agreement with epidemiologic assessments of breast cancer risk for those variants sufficiently common for case–control studies to have been undertaken. Assay results were largely concordant with consensus predictions of in silico tools, particularly for damaging alleles in the kinase domain. However, of the 25 variants, 6 were not consistently classifiable by in silico tools. An in vivo assay of cellular response to DNA damage by mutant CHEK2 alleles may complement and extend epidemiologic and genetic assessment of their clinical consequences. PMID:22419737

Response to DNA damage of CHEK2 missense mutations in familial breast cancer.

PubMed

Roeb, Wendy; Higgins, Jake; King, Mary-Claire

2012-06-15

Comprehensive sequencing of tumor suppressor genes to evaluate inherited predisposition to cancer yields many individually rare missense alleles of unknown functional and clinical consequence. To address this problem for CHEK2 missense alleles, we developed a yeast-based assay to assess in vivo CHEK2-mediated response to DNA damage. Of 25 germline CHEK2 missense alleles detected in familial breast cancer patients, 12 alleles had complete loss of DNA damage response, 8 had partial loss and 5 exhibited a DNA damage response equivalent to that mediated by wild-type CHEK2. Variants exhibiting reduced response to DNA damage were found in all domains of the CHEK2 protein. Assay results were in agreement with epidemiologic assessments of breast cancer risk for those variants sufficiently common for case-control studies to have been undertaken. Assay results were largely concordant with consensus predictions of in silico tools, particularly for damaging alleles in the kinase domain. However, of the 25 variants, 6 were not consistently classifiable by in silico tools. An in vivo assay of cellular response to DNA damage by mutant CHEK2 alleles may complement and extend epidemiologic and genetic assessment of their clinical consequences.

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay.

PubMed

Londoño-Velasco, Elizabeth; Martínez-Perafán, Fabián; Carvajal-Varona, Silvio; García-Vallejo, Felipe; Hoyos-Giraldo, Luz Stella

2016-05-01

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p <  0.001) was observed in the exposed group in comparison to the unexposed group. Neither age (%TDNA: p =  0.913) nor time of exposure (%TDNA: p = 0.398) were significantly correlated with DNA damage. The car spray painters who consumed alcohol did not show a significant increase in DNA damage compared to nonalcohol consumers (p  > 0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.

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.

Omics Approaches for Identifying Physiological Adaptations to Genome Instability in Aging.

PubMed

Edifizi, Diletta; Schumacher, Björn

2017-11-04

DNA damage causally contributes to aging and age-related diseases. The declining functioning of tissues and organs during aging can lead to the increased risk of succumbing to aging-associated diseases. Congenital syndromes that are caused by heritable mutations in DNA repair pathways lead to cancer susceptibility and accelerated aging, thus underlining the importance of genome maintenance for withstanding aging. High-throughput mass-spectrometry-based approaches have recently contributed to identifying signalling response networks and gaining a more comprehensive understanding of the physiological adaptations occurring upon unrepaired DNA damage. The insulin-like signalling pathway has been implicated in a DNA damage response (DDR) network that includes epidermal growth factor (EGF)-, AMP-activated protein kinases (AMPK)- and the target of rapamycin (TOR)-like signalling pathways, which are known regulators of growth, metabolism, and stress responses. The same pathways, together with the autophagy-mediated proteostatic response and the decline in energy metabolism have also been found to be similarly regulated during natural aging, suggesting striking parallels in the physiological adaptation upon persistent DNA damage due to DNA repair defects and long-term low-level DNA damage accumulation occurring during natural aging. These insights will be an important starting point to study the interplay between signalling networks involved in progeroid syndromes that are caused by DNA repair deficiencies and to gain new understanding of the consequences of DNA damage in the aging process.

Omics Approaches for Identifying Physiological Adaptations to Genome Instability in Aging

PubMed Central

Edifizi, Diletta; Schumacher, Björn

2017-01-01

DNA damage causally contributes to aging and age-related diseases. The declining functioning of tissues and organs during aging can lead to the increased risk of succumbing to aging-associated diseases. Congenital syndromes that are caused by heritable mutations in DNA repair pathways lead to cancer susceptibility and accelerated aging, thus underlining the importance of genome maintenance for withstanding aging. High-throughput mass-spectrometry-based approaches have recently contributed to identifying signalling response networks and gaining a more comprehensive understanding of the physiological adaptations occurring upon unrepaired DNA damage. The insulin-like signalling pathway has been implicated in a DNA damage response (DDR) network that includes epidermal growth factor (EGF)-, AMP-activated protein kinases (AMPK)- and the target of rapamycin (TOR)-like signalling pathways, which are known regulators of growth, metabolism, and stress responses. The same pathways, together with the autophagy-mediated proteostatic response and the decline in energy metabolism have also been found to be similarly regulated during natural aging, suggesting striking parallels in the physiological adaptation upon persistent DNA damage due to DNA repair defects and long-term low-level DNA damage accumulation occurring during natural aging. These insights will be an important starting point to study the interplay between signalling networks involved in progeroid syndromes that are caused by DNA repair deficiencies and to gain new understanding of the consequences of DNA damage in the aging process. PMID:29113067

The effects of male age on sperm DNA damage in healthy non-smokers.

PubMed

Schmid, T E; Eskenazi, B; Baumgartner, A; Marchetti, F; Young, S; Weldon, R; Anderson, D; Wyrobek, A J

2007-01-01

The trend for men to have children at older age 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 (mean age: 46.4 years, range: 22-80 years) with no known fertility problems using the sperm Comet analyses. The average percentage 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 under neutral conditions (P = 0.7). Men who consumed >3 cups coffee per day had approximately 20% higher percentage tail DNA under neutral but not alkaline conditions compared with men who consumed no caffeine (P = 0.005). Our findings indicate that (i) older men have increased sperm DNA damage associated with alkali-labile sites or single-strand DNA breaks and (ii) 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 of developmental defects and genetic diseases among offspring.

Archaeal RNA polymerase arrests transcription at DNA lesions.

PubMed

Gehring, Alexandra M; Santangelo, Thomas J

2017-01-01

Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

Optimised detection of mitochondrial DNA strand breaks.

PubMed

Hanna, Rebecca; Crowther, Jonathan M; Bulsara, Pallav A; Wang, Xuying; Moore, David J; Birch-Machin, Mark A

2018-05-04

Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke.

PubMed Central

Jackson, J H; Schraufstatter, I U; Hyslop, P A; Vosbeck, K; Sauerheber, R; Weitzman, S A; Cochrane, C G

1987-01-01

The mechanism by which cigarette smoking and asbestos exposure synergistically increase the incidence of lung cancer is unknown. We hypothesized that cigarette smoke and asbestos might synergistically increase DNA damage. To test this hypothesis we exposed isolated bacteriophage PM2 DNA to cigarette smoke and/or asbestos, and assessed DNA strand breaks as an index of DNA damage. Our results supported our hypothesis. 78 +/- 12% of the DNA exposed to both cigarette smoke and asbestos developed strand breaks, while only 9.8 +/- 7.0 or 4.3 +/- 3.3% of the DNA exposed to cigarette smoke or asbestos, respectively, developed strand breaks under the conditions of the experiment. Our experimental evidence suggested that cigarette smoke and asbestos synergistically increased DNA damage by stimulating .OH formation. First, significant amounts of .OH were detected by electron paramagnetic resonance (EPR) in DNA mixtures containing both cigarette smoke and asbestos, but no .OH was detected in mixtures containing cigarette smoke alone or asbestos alone. Second, the .OH scavengers, dimethylsulfoxide (DMSO), mannitol, or Na benzoate decreased both .OH detection by EPR and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. Third, the H2O2 scavenger, catalase, and the iron chelators, 1,10-phenanthroline and desferrithiocin, decreased both .OH detection and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. These latter findings suggest that iron contained in asbestos may catalyze the formation of .OH from H2O2 generated by cigarette smoke. In summary, our study indicates that cigarette smoke and asbestos synergistically increase DNA damage and suggests that this synergism may involve .OH production. PMID:2821073

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

PubMed Central

Klapacz, Joanna; Pottenger, Lynn H.; Engelward, Bevin P.; Heinen, Christopher D.; Johnson, George E.; Clewell, Rebecca A.; Carmichael, Paul L.; Adeleye, Yeyejide; Andersen, Melvin E.

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

PubMed

Klapacz, Joanna; Pottenger, Lynn H; Engelward, Bevin P; Heinen, Christopher D; Johnson, George E; Clewell, Rebecca A; Carmichael, Paul L; Adeleye, Yeyejide; Andersen, Melvin E

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultraviolet-B-induced DNA damage and ultraviolet-B tolerance mechanisms in species with different functional groups coexisting in subalpine moorlands.

PubMed

Wang, Qing-Wei; Kamiyama, Chiho; Hidema, Jun; Hikosaka, Kouki

2016-08-01

High doses of ultraviolet-B (UV-B; 280-315 nm) radiation can have detrimental effects on plants, and especially damage their DNA. Plants have DNA repair and protection mechanisms to prevent UV-B damage. However, it remains unclear how DNA damage and tolerance mechanisms vary among field species. We studied DNA damage and tolerance mechanisms in 26 species with different functional groups coexisting in two moorlands at two elevations. We collected current-year leaves in July and August, and determined accumulation of cyclobutane pyrimidine dimer (CPD) as UV-B damage and photorepair activity (PRA) and concentrations of UV-absorbing compounds (UACs) and carotenoids (CARs) as UV-B tolerance mechanisms. DNA damage was greater in dicot than in monocot species, and higher in herbaceous than in woody species. Evergreen species accumulated more CPDs than deciduous species. PRA was higher in Poaceae than in species of other families. UACs were significantly higher in woody than in herbaceous species. The CPD level was not explained by the mechanisms across species, but was significantly related to PRA and UACs when we ignored species with low CPD, PRA and UACs, implying the presence of another effective tolerance mechanism. UACs were correlated negatively with PRA and positively with CARs. Our results revealed that UV-induced DNA damage significantly varies among native species, and this variation is related to functional groups. DNA repair, rather than UV-B protection, dominates in UV-B tolerance in the field. Our findings also suggest that UV-B tolerance mechanisms vary among species under evolutionary trade-off and synergism.

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.

PubMed

Ganesan, Shanthi; Keating, Aileen F

2015-02-01

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 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h 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. Copyright © 2014 Elsevier Inc. All rights reserved.

Association of HSP70 and genotoxic damage in lymphocytes of workers exposed to coke-oven emission

PubMed Central

Xiao, Chengfeng; Chen, Sheng; Li, Jizhao; Hai, Tao; Lu, Qiaofa; Sun, Enling; Wang, Ruibo; Tanguay, Robert M.; Wu, Tangchun

2002-01-01

Heat shock proteins (Hsps) have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli. Whether they protect against deoxyribonucleic acid (DNA) damage in individuals exposed to environmental stresses and chemical carcinogens is unknown. In the study, we investigated the association between Hsp70 levels (the most abundant mammalian Hsp) and genotoxic damage in lymphocytes of workers exposed to coke-oven emission using Western dot blot and 2 DNA damage assays, the comet assay and the micronucleus test. The data show that there is a significant increase in Hsp70 levels, DNA damage score, and micronucleus rates in lymphocytes of workers exposed to coke-oven emission as compared with the control subjects. Furthermore, there was a significant negative correlation of Hsp70 levels with DNA damage scores in the comet assay (r = −0.663, P < 0.01) and with micronucleus rates (r = −0.461, P < 0.01) in the exposed group. In the control group, there was also a light negative correlation between Hsp70 with DNA damage and micronuclei rate (r = −0.236 and r = 0.242, respectively), but it did not reach a statistically significant level (P > 0.05). Our results show that individuals who had high Hsp70 levels generally showed lower genotoxic damage than others. These results suggest a role of Hsp70 in the protection of DNA from genotoxic damage induced by coke-oven emission. PMID:12653484

Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river

USGS Publications Warehouse

Karouna-Renier, Natalie K.; White, Carl; Perkins, Christopher R.; Schmerfeld, John J.; Yates, David

2014-01-01

Historical discharges of Hg into the South River near the town of Waynesboro, VA, USA, have resulted in persistently elevated Hg concentrations in sediment, surface water, ground water, soil, and wildlife downstream of the discharge site. In the present study, we examined mercury (Hg) levels in in little brown bats (Myotis lucifugus) from this location and assessed the utility of a non-destructively collected tissue sample (wing punch) for determining mitochondrial DNA (mtDNA) damage in Hg exposed bats. Bats captured 1 and 3 km from the South River, exhibited significantly higher levels of total Hg (THg) in blood and fur than those from the reference location. We compared levels of mtDNA damage using real-time quantitative PCR (qPCR) analysis of two distinct regions of mtDNA. Genotoxicity is among the many known toxic effects of Hg, resulting from direct interactions with DNA or from oxidative damage. Because it lacks many of the protective protein structures and repair mechanisms associated with nuclear DNA, mtDNA is more sensitive to the effects of genotoxic chemicals and therefore may be a useful biomarker in chronically exposed organisms. Significantly higher levels of damage were observed in both regions of mtDNA in bats captured 3 km from the river than in controls. However, levels of mtDNA damage exhibited weak correlations with fur and blood THg levels, suggesting that other factors may play a role in the site-specific differences.

77 FR 18833 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-28

... mass spectra obtained and reproduced for food-borne pathogens. Unique DISI device with gas cylinder... With a Small Molecule CHK2 Inhibitor Description of Technology: DNA damage sensors such as Checkpoint... in response to DNA damage. It has been reported that these DNA damage sensors also play a key role in...

DNA Damage Repair Factors have a Tumor Promoting Role in MLL-fusion Leukemia | Center for Cancer Research

Cancer.gov

Cancers develop when cells accumulate DNA mutations that allow them to grow and divide inappropriately. Thus, proteins involved in repairing DNA damage are generally suppressors of cancer formation, and their expression is often lost in the early stages of cancer initiation. In contrast, cancer stem cells, like their normal counterparts, must retain their ability to self-renew, which necessitates maintenance of DNA integrity. In hematopoietic stem cells (HSC), for example, double strand breaks and oxidative damage exhaust their regenerative ability. André Nussenzweig, Ph.D., Chief of CCR’s Laboratory of Genome Integrity and his colleagues wondered whether leukemic stem cells might be similarly constrained by DNA damage.

DNA Damage and Repair in Plants under Ultraviolet and Ionizing Radiations

PubMed Central

Gill, Sarvajeet S.; 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. PMID:25729769

Characteristics and oxidative stress on rats and traffic policemen of ambient fine particulate matter from Shenyang.

PubMed

Ma, Mingyue; Li, Shuyin; Jin, Huanrong; Zhang, Yumin; Xu, Jia; Chen, Dongmei; Kuimin, Chen; Yuan, Zhou; Xiao, Chunling

2015-09-01

Fine particulate matter (PM2.5) pollution is becoming serious in China. This study aimed to investigate the impact of PM2.5 on DNA damage in Shenyang city. The concentration and composition of PM2.5 in traffic policemen's working sites including fields and indoor offices were obtained. Blood samples of field and office policemen were collected to detect DNA damage by Comet assay. Rats were used to further analyzing the oxidative DNA damage. The average concentration of PM2.5 in exposed group was significantly higher than that in control group. Composition analysis revealed that toxic heavy metal and polycyclic aromatic hydrocarbon substances were main elements of this PM2.5. DNA damage in field policemen was significantly higher than those in non-field group. Moreover, animal studies confirmed the oxidative DNA damage induced by PM2.5. Taken together, high DNA damages are found in the Shenyang traffic policemen and rats exposed to high level of airborne PM2.5. Copyright © 2015 Elsevier B.V. All rights reserved.

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway.

PubMed

Llanos, Susana; Serrano, Manuel

2010-10-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.

Editorial Commentary: "Defer No Time, Delays Have Dangerous Ends" (Henry VI, Shakespeare): Delayed Anterior Cruciate Ligament Reconstruction Has Consequences.

PubMed

Siegel, Mark G

2018-06-01

There continues to be controversy over the timing of anterior cruciate ligament (ACL) surgery. Early or delayed intervention after ACL injury is a topic that has not been settled. The issue is whether ACL tears should have surgery performed in an expedient manner. Or is delay an option with no repercussions to the health of the knee? My associates in nonsurgical specialties wave the New England Journal of Medicine to support their view that surgery is not needed. I routinely espouse the literature confirming that delay of surgery may cause future damage. It is now established that a failure to intervene in a timely manner does cause additional damage. I stand vindicated and can affirm to my colleagues that I have found the answer. There is no longer any doubt or equivocation. Delay in reconstructing an unstable knee does cause damage. Copyright © 2018 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

The sequence specificity of UV-induced DNA damage in a systematically altered DNA sequence.

PubMed

Khoe, Clairine V; Chung, Long H; Murray, Vincent

2018-06-01

The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

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

Dong, Hui; Shi, Qiong; Song, Xiufang

2015-07-01

Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observedmore » phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.« less

Importance of cell damage causing growth delay for high pressure inactivation of Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Nanba, Masaru; Nomura, Kazuki; Nasuhara, Yusuke; Hayashi, Manabu; Kido, Miyuki; Hayashi, Mayumi; Iguchi, Akinori; Shigematsu, Toru; Hirayama, Masao; Ueno, Shigeaki; Fujii, Tomoyuki

2013-06-01

A high pressure (HP) tolerant (barotolerant) mutant a2568D8 and a variably barotolerant mutant a1210H12 were generated from Saccharomyces cerevisiae using ultra-violet mutagenesis. The two mutants, a barosensitive mutant a924E1 and the wild-type strain, were pressurized (225 MPa), and pressure inactivation behavior was analyzed. In the wild-type strain, a proportion of the growth-delayed cells were detected after exposure to HP. In a924E1, the proportion of growth-delayed cells significantly decreased compared with the wild-type. In a2568D8, the proportion of growth-delayed cells increased and the proportion of inactivated cells decreased compared with the wild-type. In a1210H12, the growth-delayed cells could not be detected within 120 s of exposure to HP. The proportion of growth-delayed cells, which incurred the damage, would affect the survival ratio by HP. These results suggested that cellular changes in barotolerance caused by mutations are remarkably affected by the ability to recover from cellular damage, which results in a growth delay.

Assessment of genotoxic effects of flumorph by the comet assay in mice organs.

PubMed

Zhang, T; Zhao, Q; Zhang, Y; Ning, J

2014-03-01

The present study investigated the genotoxic effects of flumorph in various organs (brain, liver, spleen, kidney and sperm) of mice. The DNA damage, measured as comet tail length (µm), was determined using the alkaline comet assay. The comet assay is a sensitive assay for the detection of genotoxicity caused by flumorph using mice as a model. Statistically significant increases in comet assay for both dose-dependent and duration-dependent DNA damage were observed in all the organs assessed. The organs exhibited the maximum DNA damage in 96 h at 54 mg/kg body weight. Brain showed maximum DNA damage followed by spleen > kidney > liver > sperm. Our data demonstrated that flumorph had induced systemic genotoxicity in mammals as it caused DNA damage in all tested vital organs, especially in brain and spleen.