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Sample records for atr-dependent dna damage

  1. Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells.

    PubMed

    Jang, Sung Hee; Lim, Joo Weon; Morio, Tomohiro; Kim, Hyeyoung

    2012-02-01

    Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.

  2. ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function

    PubMed Central

    Collins, Natalie B.; Wilson, James B.; Bush, Thomas; Thomashevski, Andrei; Roberts, Kate J.; Jones, Nigel J.

    2009-01-01

    Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway. PMID:19109555

  3. Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*

    PubMed Central

    Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl

    2015-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis). PMID:25561503

  4. Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway.

    PubMed

    Rizzo, Angela; Salvati, Erica; Porru, Manuela; D'Angelo, Carmen; Stevens, Malcolm F; D'Incalci, Maurizio; Leonetti, Carlo; Gilson, Eric; Zupi, Gabriella; Biroccio, Annamaria

    2009-09-01

    Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.

  5. Loss of Smu1 function de-represses DNA replication and over-activates ATR-dependent replication checkpoint.

    PubMed

    Ren, Laifeng; Liu, Yao; Guo, Liandi; Wang, Haibin; Ma, Lei; Zeng, Ming; Shao, Xin; Yang, Chunlei; Tang, Yaxiong; Wang, Lei; Liu, Cong; Li, Mingyuan

    2013-06-28

    Smu1 is an evolutionarily conserved gene that encodes a member of the WD40-repeat protein family. Disruption of Smu1 function leads to multiple cellular defects including chromosomal instability, aberrant DNA replication and alternative RNA splicing events. In this paper, we show that Smu1 is a chromatin-bound protein that functions as a negative regulator of DNA replication. Knockdown of Smu1 gene expression promotes excessive incorporation of dNTP analogue, implicating the acceleration of DNA synthesis. Smu1-silenced cells show an excessive activation of replication checkpoint in response to ultraviolate (UV) or hydroxyurea treatment, indicating that abnormal stimulation of DNA replication leads to instability of genomic structure. Hence, we propose that Smu1 participates in the protection of genomic integrity by negatively regulating the process of DNA synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. DNA damage response and transcription.

    PubMed

    Lagerwerf, Saskia; Vrouwe, Mischa G; Overmeer, René M; Fousteri, Maria I; Mullenders, Leon H F

    2011-07-15

    A network of DNA damage surveillance systems is triggered by sensing of DNA lesions and the initiation of a signal transduction cascade that activates genome-protection pathways including nucleotide excision repair (NER). NER operates through coordinated assembly of repair factors into pre- and post-incision complexes. Recent work identifies RPA as a key regulator of the transition from dual incision to repair-synthesis in UV-irradiated non-cycling cells, thereby averting the generation of unprocessed repair intermediates. These intermediates could lead to recombinogenic events and trigger a persistent ATR-dependent checkpoint signaling. It is now evident that DNA damage signaling is not limited to NER proficient cells. ATR-dependent checkpoint activation also occurs in UV-exposed non-cycling repair deficient cells coinciding with the formation of endonuclease APE1-mediated DNA strand breaks. In addition, the encounter of elongating RNA polymerase II (RNAPIIo) with DNA damage lesions and its persistent stalling provides a strong DNA damage signaling leading to cell cycle arrest, apoptosis and increased mutagenesis. The mechanism underlying the strong and strand specific induction of UV-induced mutations in NER deficient cells has been recently resolved by the finding that gene transcription itself increases UV-induced mutagenesis in a strand specific manner via increased deamination of cytosines. The cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER) without displacement of the DNA damage stalled RNAPIIo. Deficiency in TC-NER associates with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). CSB functions as a repair coupling factor to attract NER proteins, chromatin remodelers and the CSA-E3-ubiquitin ligase complex to the stalled RNAPIIo; CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1

  7. DNA Double-Strand Breaks by Cr(VI) Are Targeted to Euchromatin and Cause ATR-Dependent Phosphorylation of Histone H2AX and Its Ubiquitination

    PubMed Central

    DeLoughery, Zachary; Luczak, Michal W.; Ortega-Atienza, Sara; Zhitkovich, Anatoly

    2015-01-01

    Hexavalent chromium is a human respiratory carcinogen that undergoes intracellular activation in vivo primarily via reduction with ascorbate. Replication of Cr-adducted DNA triggers mismatch repair that generates toxic DNA double-strand breaks (DSBs) as secondary lesions. Here, we examined the intranuclear distribution of chromate-induced breaks and a central DSB signaling branch targeting histone H2AX. Using ascorbate-restored cells (H460 human lung epithelial cells, normal human lung and normal mouse embryonic fibroblasts (MEFs)), we found that Cr(VI) produced a typical DSB-associated spectrum of H2AX modifications, including its Ser139-phosphorylated (known as γH2AX) and mono- and diubiquitinated forms. However, whereas canonical DSB signaling relies on ATM, the formation of γH2AX and its ubiquitinated products by Cr(VI) was dependent on ATR kinase. Based on the established mode of ATR activation, this suggests that Cr-induced DSB are not blunt-ended and likely contain single-stranded tails. Confocal imaging with markers of active and inactive chromatin revealed a selective formation of Cr-induced DSB in euchromatin of mouse and human cells. In contrast to DSB, Cr-DNA adducts were produced in both types of chromatin. The euchromatin targeting of Cr-induced DSB makes these lesions particularly dangerous by increasing the probability of deleting active tumor suppressors and producing oncogenic translocations. Accumulation of transcription-inhibiting ubiquitinated forms of γH2AX in euchromatin is expected to contribute to the ability of Cr(VI) to suppress upregulation of inducible genes. PMID:25288669

  8. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  9. A novel role for Greatwall kinase in recovery from DNA damage

    PubMed Central

    Yamamoto, Tomomi M; Goldberg, Michael L

    2010-01-01

    Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression. The shut off process of the DDR upon satisfaction of DNA repair, also known as “checkpoint recovery,” involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition and helps maintain M phase through inhibition of PP2A/B55δ, the principal phosphatase for Cdk-phosphorylated substrates. Here, we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild-type, but not kinase-dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl overexpression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA damage. PMID:20980823

  10. ATM/ATR-dependent Tyr15 phosphorylation of cyclin-dependent kinases in response to hydroxyurea in Vicia faba root meristem cells.

    PubMed

    Winnicki, Konrad

    2013-10-01

    DNA damage or stalled replication forks activate cell cycle checkpoints. However, the regulation of metabolic pathways that are responsible for maintenance of genome integrity in plants is still largely unknown. Present research on Vicia faba root meristem cells indicates that inhibitory phosphorylation of cyclin-dependent kinases (Cdks) at Tyr15 plays a prominent role during blockage of cell cycle in response to genotoxic stress. Phosphorylation of P-loop in Cdks takes place in ATM/ATR-dependent manner. Although, Tyr15 phosphorylation upon hydroxyurea (HU) treatment was found in most cells classified to G1 and S phase, interestingly, the number of phoshpo-Tyr15-positive cells decreases in G2 phase. Presented data confirm much similarity in regulation of Cdks functions under genotoxic stress between plants and animals; however, they may also substantiate evolutionarily developed differences especially in regulation of G2/M transition between these two kingdoms.

  11. DNA damage and mutation. Types of DNA damage

    NASA Astrophysics Data System (ADS)

    Chakarov, Stoyan; Petkova, Rumena; Russev, George Ch; Zhelev, Nikolai

    2014-02-01

    This review outlines the basic types of DNA damage caused by exogenous and endogenous factors, analyses the possible consequences of each type of damage and discusses the need for different types of DNA repair. The mechanisms by which a minor damaging event to DNA may eventually result in the introduction of heritable mutation/s are reviewed. The major features of the role of DNA damage in ageing and carcinogenesis are outlined and the role of iatrogenic DNA damage in human health and disease (with curative intent as well as a long-term adverse effect of genotoxic therapies) are discussed in detail.

  12. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  13. Ribonucleotide triggered DNA damage and RNA-DNA damage responses.

    PubMed

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.

  14. DNA damage response defect in Williams-Beuren syndrome

    PubMed Central

    Guenat, David; Merla, Giuseppe; Deconinck, Eric; Borg, Christophe; Rohrlich, Pierre-Simon

    2017-01-01

    Williams-Beuren syndrome (WBS, no. OMIM 194050) is a rare multisystem genetic disorder caused by a microdeletion on chromosome 7q11.23 and characterized by cardiovascular malformations, mental retardation, and a specific facial dysmorphism. Recently, we reported that a series of non-Hodgkin's lymphoma occurs in children with WBS and thus hypothesized that a predisposition to cancer may be associated with this genetic disorder. The aim of the present study was to ascertain the role played by three genes hemizygously deleted in WBS (RFC2, GTF2I and BAZ1B) in DNA damage response pathways. Cell proliferation, cell cycle analysis, γ-H2A.X induction, and expression of DNA damage response proteins were investigated upon exposure to genotoxic treatments in WBS patient-derived primary fibroblasts and in the 293T cell line treated with specific siRNAs targeting RFC2, GTF2I and BAZ1B. An impaired hydroxyurea-induced phosphorylation of CHK1 was observed in the WBS cells. However, this defective DNA damage response was not associated with an increased sensitivity to genotoxic agents. In addition, depletion of RFC2, GTF2I and BAZ1B using specific siRNAs did not have a significant impact on the DNA damage response in 293T cells. Our results highlight that the ATR-dependent DNA damage response is impaired in WBS patient cells but is also dispensable for viability when these cells undergo a genotoxic stress. The mechanism by which the ATR pathway is impaired in WBS warrants elucidation through further investigation. PMID:28098859

  15. DNA damage and breast cancer

    PubMed Central

    Davis, Jennifer D; Lin, Shiaw-Yih

    2011-01-01

    Cancer is intimately related to the accumulation of DNA damage, and repair failures (including mutation prone repair and hyperactive repair systems). This article relates current clinical categories for breast cancer and their common DNA damage repair defects. Information is included on the potential for accumulation of DNA damage in the breast tissue of a woman during her lifetime and the role of DNA damage in breast cancer development. We then cover endogenous and exogenous sources of DNA damage, types of DNA damage repair and basic signal transduction pathways for three gene products involved in the DNA damage response system; namely BRCA1, BRIT1 and PARP-1. These genes are often considered tumor suppressors because of their roles in DNA damage response and some are under clinical investigation as likely sources for effective new drugs to treat breast cancers. Finally we discuss some of the problems of DNA damage repair systems in cancer and the conundrum of hyper-active repair systems which can introduce mutations and confer a survival advantage to certain types of cancer cells. PMID:21909479

  16. DNA Damage, DNA Repair, Aging, and Neurodegeneration.

    PubMed

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

    2015-09-18

    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.

  17. 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

  18. DNA Damage Induced Neuronal Death

    DTIC Science & Technology

    1999-10-01

    Experiments are proposed to examine the molecular mechanism by which mustard chemical warfare agents induce neuronal cell death . DNA damage is the...proposed underlying mechanism of mustard-induced neuronal cell death . We propose a novel research strategy to test this hypothesis by using mice with...perturbed DNA repair to explore the relationship between mustard-induced DNA damage and neuronal cell death . Initial in vitro studies (Years 1, 2 & 3

  19. Function of ZFAND3 in the DNA Damage Response

    DTIC Science & Technology

    2012-06-01

    treatment with various genotoxic agents was examined. Signaling from the apical DDR kinase ATR (ataxia telangiectasia- mutated ( ATM ) and Rad3...to ATR- dependent checkpoint signaling after IR. It remains possible that the knock-down Figure 2. ZFAND3 silencing does not hypersensitize...resolution achieved with iPOND depends on EdU incubation time, the rate of DNA synthesis and chromatin fragment size. Experimentally, EdU incubation

  20. Optical detection of DNA damage

    NASA Astrophysics Data System (ADS)

    Rogers, Kim R.; Apostol, A.; Cembrano, J.

    1999-02-01

    A rapid and sensitive fluorescence assay for oxidative damage to calf thymus DNA is reported. A decrease in the transition temperature for strand separation resulted from exposure of the DNA to the reactive decomposition products of 3- morpholinosydnonimine (SIN-1) (i.e., nitric oxide, superoxide, peroxynitrite, hydrogen peroxide, and hydroxyl radicals). A decrease in melting temperature of 12 degrees Celsius was indicative of oxidative damage including single strand chain breaks. Double stranded (ds) and single stranded (ss) forms of DNA were determined using the indicator dyes ethidium bromide and PicoGreen. The change in DNA 'melting' curves was dependant on the concentration of SIN-1 and was most pronounced at 75 degrees Celsius. This chemically induced damage was significantly inhibited by sodium citrate, tris(hydroxymethyl)aminomethane (Tris), and diethylenetriaminepentaacetic acid (DTPA), but was unaffected by superoxide dismutase (SOD), catalase, ethylenediamine tetraacietic acid (EDTA), or deferoxamine. Lowest observable effect level for SIN-1-induced damage was 200 (mu) M.

  1. Deciphering the DNA Damage Response.

    PubMed

    Haber, James E

    2015-09-10

    This year's Albert Lasker Basic Medical Research Award honors Evelyn Witkin and Stephen J. Elledge, two pioneers in elucidating the DNA damage response, whose contributions span more than 40 years. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Replicating damaged DNA in eukaryotes.

    PubMed

    Chatterjee, Nimrat; Siede, Wolfram

    2013-12-01

    DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.

  3. DNA Repair by Reversal of DNA Damage

    PubMed Central

    Yi, Chengqi; He, Chuan

    2013-01-01

    Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology. PMID:23284047

  4. DNA damage and carcinogenesis

    SciTech Connect

    Stelow, R B

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10/sup 4/ fold.

  5. Sperm DNA oxidative damage and DNA adducts

    PubMed Central

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-01-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

  6. Haploinsufficiency of DNA Damage Response Genes and their Potential Influence in Human Genomic Disorders

    PubMed Central

    O’Driscoll, Mark

    2008-01-01

    Genomic disorders are a clinically diverse group of conditions caused by gain, loss or re-orientation of a genomic region containing dosage-sensitive genes. One class of genomic disorder is caused by hemizygous deletions resulting in haploinsufficiency of a single or, more usually, several genes. For example, the heterozygous contiguous gene deletion on chromosome 22q11.2 causing DiGeorge syndrome involves at least 20-30 genes. Determining how the copy number variation (CNV) affects human variation and contributes to the aetiology and progression of various genomic disorders represents important questions for the future. Here, I will discuss the functional significance of one form of CNV, haploinsufficiency (i.e. loss of a gene copy), of DNA damage response components and its association with certain genomic disorders. There is increasing evidence that haploinsufficiency for certain genes encoding key players in the cells response to DNA damage, particularly those of the Ataxia Telangiectasia and Rad3-related (ATR)-pathway, has a functional impact. I will review this evidence and present examples of some well known clinically similar genomic disorders that have recently been shown to be defective in the ATR-dependent DNA damage response. Finally, I will discuss the potential implications of a haploinsufficiency-induced defective DNA damage response for the clinical management of certain human genomic disorders. PMID:19440510

  7. Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

    PubMed

    Horvath, Beatrix M; Kourova, Hana; Nagy, Szilvia; Nemeth, Edit; Magyar, Zoltan; Papdi, Csaba; Ahmad, Zaki; Sanchez-Perez, Gabino F; Perilli, Serena; Blilou, Ikram; Pettkó-Szandtner, Aladár; Darula, Zsuzsanna; Meszaros, Tamas; Binarova, Pavla; Bogre, Laszlo; Scheres, Ben

    2017-05-02

    The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  8. 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...

  9. 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...

  10. DNA Damage and Pulmonary Hypertension.

    PubMed

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-06-22

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis.

  11. DNA Damage and Pulmonary Hypertension

    PubMed Central

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  12. Modulation of the DNA damage response during the life cycle of human papillomaviruses.

    PubMed

    Anacker, Daniel C; Moody, Cary A

    2017-03-02

    Human papillomavirus (HPV) is the most common sexually transmitted viral infection. Infection with certain types of HPV pose a major public health risk as these types are associated with multiple human cancers, including cervical cancer, other anogenital malignancies and an increasing number of head and neck cancers. The HPV life cycle is closely tied to host cell differentiation with late viral events such as structural gene expression and viral genome amplification taking place in the upper layers of the stratified epithelium. The DNA damage response (DDR) is an elaborate signaling network of proteins that regulate the fidelity of replication by detecting, signaling and repairing DNA lesions. ATM and ATR are two kinases that are major regulators of DNA damage detection and repair. A multitude of studies indicate that activation of the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3-related) pathways are critical for HPV to productively replicate. This review outlines how HPV interfaces with the ATM- and ATR-dependent DNA damage responses throughout the viral life cycle to create an environment supportive of viral replication and how activation of these pathways could impact genomic stability. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. DNA Damage and Repair in Vascular Disease.

    PubMed

    Uryga, Anna; Gray, Kelly; Bennett, Martin

    2016-01-01

    DNA damage affecting both genomic and mitochondrial DNA is present in a variety of both inherited and acquired vascular diseases. Multiple cell types show persistent DNA damage and a range of lesions. In turn, DNA damage activates a variety of DNA repair mechanisms, many of which are activated in vascular disease. Such DNA repair mechanisms either stall the cell cycle to allow repair to occur or trigger apoptosis or cell senescence to prevent propagation of damaged DNA. Recent evidence has indicated that DNA damage occurs early, is progressive, and is sufficient to impair function of cells composing the vascular wall. The consequences of persistent genomic and mitochondrial DNA damage, including inflammation, cell senescence, and apoptosis, are present in vascular disease. DNA damage can thus directly cause vascular disease, opening up new possibilities for both prevention and treatment. We review the evidence for and the causes, types, and consequences of DNA damage in vascular disease.

  14. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

  15. 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.

  16. DNA damage by various radiations

    NASA Astrophysics Data System (ADS)

    Hasegawa, K.; Yoshioka, H.; Yoshioka, H.

    1997-01-01

    In an attempt to shed light on the influence of tritiated water on DNA we have investigated the post-irradiation damage with a simple plasmid DNA, pBR322 and pUC18. The survival of covalently closed circular (CCC) DNA form was directly followed by agarose gel electrophoresis. The survival percentage of DNA in tritiated water was almost the same as with the irradiation with X-rays at the same absorbed dose. For irradiation with γ-rays, on the other hand, the decay rate was larger than those observed with both tritiated water and X-rays. The percentages of breakage for DNA in tritiated water, X-rays and γ-rays were found to be 34, 38 and 33% at 100 Gy of absorbed dose. The effect of dose rate was not observed for irradiation with tritiated water, X-rays and γ-rays. In order to study protection of DNA against radiation, we investigated the protecting effect of tea catechin which is the main component of (-)-epigallocatechin gallate (EGCg). The protection mechanism for DNA against radiation-induced scission has been studied using ESR spin-trapping method.

  17. Could Shift Work Damage Your DNA?

    MedlinePlus

    ... fullstory_166904.html Could Shift Work Damage Your DNA? Small study raises questions about daytime sleep and ... less capacity to repair everyday damage to cells' DNA, a small study hints. The research found that ...

  18. Sphingolipids in the DNA damage response.

    PubMed

    Carroll, Brittany; Donaldson, Jane Catalina; Obeid, Lina

    2015-05-01

    Recently, sphingolipid metabolizing enzymes have emerged as important targets of many chemotherapeutics and DNA damaging agents and therefore play significant roles in mediating the physiological response of the cell to DNA damage. In this review we will highlight points of connection between the DNA damage response (DDR) and sphingolipid metabolism; specifically how certain sphingolipid enzymes are regulated in response to DNA damage and how the bioactive lipids produced by these enzymes affect cell fate. Published by Elsevier Ltd.

  19. Sphingolipids in the DNA Damage Response

    PubMed Central

    Carroll, Brittany; Donaldson, Cat; Obeid, Lina

    2014-01-01

    Recently, sphingolipid metabolizing enzymes have emerged as important targets of many chemotherapeutics and DNA damaging agents and therefore play significant roles in mediating the physiological response of the cell to DNA damage. In this review we will highlight points of connection between the DNA damage response (DDR) and sphingolipid metabolism; specifically how certain sphingolipid enzymes are regulated in response to DNA damage and how the bioactive lipids produced by these enzymes affect cell fate. PMID:25434743

  20. p27kip1 stabilization is essential for the maintenance of cell cycle arrest in response to DNA damage

    PubMed Central

    Cuadrado, Myriam; Gutierrez-Martinez, Paula; Swat, Aneta; Nebreda, Angel R.; Fernandez-Capetillo, Oscar

    2013-01-01

    One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27kip1 and retinoblastoma tumour suppressors (TS), and which is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance –but not the initiation- of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ATM- and ATR-dependent DDR, but downstream of p38 MAPK. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important TSs in human cancer. PMID:19843869

  1. USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

    PubMed Central

    Kim, Jihoon; D'Annibale, Sara; Magliozzi, Roberto; Low, Teck Yew; Jansen, Petra; Shaltiel, Indra A.; Mohammed, Shabaz; Heck, Albert J. R.; Medema, Rene H.

    2014-01-01

    In response to genotoxic stress, DNA damage checkpoints maintain the integrity of the genome by delaying cell cycle progression to allow for DNA repair. Here we show that the degradation of the basic helix-loop-helix (bHLH) transcription factor DEC1, a critical regulator of cell fate and circadian rhythms, controls the DNA damage response. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by the SCFβTrCP ubiquitin ligase in cooperation with CK1. Upon DNA damage, DEC1 is rapidly induced in an ATM/ATR-dependent manner. DEC1 induction results from protein stabilization via a mechanism that requires the USP17 ubiquitin protease. USP17 binds and deubiquitylates DEC1, markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 proteolysis is reestablished through βTrCP-dependent ubiquitylation. Expression of a degradation-resistant DEC1 mutant prevents checkpoint recovery by inhibiting the downregulation of p53. These results indicate that the regulated degradation of DEC1 is a key factor controlling the DNA damage response. PMID:25202122

  2. Interactions between DNA damage, repair, and transcription.

    PubMed

    Khobta, Andriy; Epe, Bernd

    2012-08-01

    This review addresses a variety of mechanisms by which DNA repair interacts with transcription and vice versa. Blocking of transcriptional elongation is the best studied of these mechanisms. Transcription recovery after damage therefore has often been used as a surrogate marker of DNA repair in cells. However, it has become evident that relationships between DNA damage, repair, and transcription are more complex due to various indirect effects of DNA damage on gene transcription. These include inhibition of transcription by DNA repair intermediates as well as regulation of transcription and of the epigenetic status of the genes by DNA repair-related mechanisms. In addition, since transcription is emerging as an important endogenous source of DNA damage in cells, we briefly summarise recent advances in understanding the nature of co-transcriptionally induced DNA damage and the DNA repair pathways involved. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Electrochemical DNA Sensors for Detection of DNA Damage

    PubMed Central

    Diculescu, Victor Constantin; Paquim, Ana-Maria Chiorcea; Brett, Ana Maria Oliveira

    2005-01-01

    Electrochemical devices have received particular attention due to their rapid detection and great sensitivity for the evaluation of DNA-hazard compounds interaction mechanisms. Several types of bioanalytical method use nucleic acids probes to detect DNA damage. This article reviews current directions and strategies in the development and applications of electrochemical DNA sensors for the detection of DNA damage.

  4. Rapid Approaches Towards DNA Damage Analysis

    PubMed Central

    Crews, N.; Paidipalli, Manasa

    2013-01-01

    The severe effects of DNA damage on human health have led to a tremendous amount of research being focused. Owing to the importance of damage detection, different approaches for the detection and quantification of the damaged DNA will be presented. In this work, we have modeled DNA damage using well-known mutagens: UV radiation to create photoproducts and restriction enzyme digestion to create double strand breaks. We will show that quantitative PCR (qPCR), a widely known measure of detecting the presence of the target DNA can be used to quantify photoproducts/intramolecular DNA damage. Our results indicate that a comparison of the initial concentration available in the undamaged and the damaged samples can be used to reveal the effect of damaged DNA in its amplification. By analyzing multiple regions using this technique, their relative susceptibility to damage can be measured. We also show that high-resolution melting analysis (HRMA), a measure of the bond energy between two DNA strands, can be used to quantify double strand breaks. The strand breaks resulted in a change in the overall distribution of the bond energy thus causing variations in the melting profile. HRMA has also been examined to compare DNA damage resulting from UV-A, UV-B and UV-C irradiation. The evaluation techniques demonstrated can potentially be extended to various types of DNA damage.

  5. Histone modifications in DNA damage response.

    PubMed

    Cao, Lin-Lin; Shen, Changchun; Zhu, Wei-Guo

    2016-03-01

    DNA damage is a relatively common event in eukaryotic cell and may lead to genetic mutation and even cancer. DNA damage induces cellular responses that enable the cell either to repair the damaged DNA or cope with the damage in an appropriate way. Histone proteins are also the fundamental building blocks of eukaryotic chromatin besides DNA, and many types of post-translational modifications often occur on tails of histones. Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in several chromatin-based processes, such as DNA damage response. In this review, we will discuss the main histone modifications, and their functions in DNA damage response.

  6. Using DNA damage to monitor water environment

    NASA Astrophysics Data System (ADS)

    Zhu, Liyan; Huang, Ying; Liu, Guangxing

    2005-09-01

    DNA damage of aquatic organisms living in polluted environments can be used as a biomarker of the genotoxicity of toxic agents to organisms. This technique has been playing an important role in ecotoxicological study and environmental risk assessment. In this article, main types of DNA damage caused by pollutants in water environments were reviewed; methods of detecting DNA damage were also documented for water environmental monitoring.

  7. DNA damage response, redox status and hematopoiesis.

    PubMed

    Weiss, Cary N; Ito, Keisuke

    2014-01-01

    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.

  8. Oxidative DNA damage & repair: An introduction.

    PubMed

    Cadet, Jean; Davies, Kelvin J A

    2017-06-01

    This introductory article should be viewed as a prologue to the Free Radical Biology & Medicine Special Issue devoted to the important topic of Oxidatively Damaged DNA and its Repair. This special issue is dedicated to Professor Tomas Lindahl, co-winner of the 2015 Nobel Prize in Chemistry for his seminal discoveries in the area repair of oxidatively damaged DNA. In the past several years it has become abundantly clear that DNA oxidation is a major consequence of life in an oxygen-rich environment. Concomitantly, survival in the presence of oxygen, with the constant threat of deleterious DNA mutations and deletions, has largely been made possible through the evolution of a vast array of DNA repair enzymes. The articles in this Oxidatively Damaged DNA & Repair special issue detail the reactions by which intracellular DNA is oxidatively damaged, and the enzymatic reactions and pathways by which living organisms survive such assaults by repair processes. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. DNA damage signalling prevents deleterious telomere addition at DNA breaks

    PubMed Central

    Makovets, Svetlana; Blackburn, Elizabeth H.

    2009-01-01

    The response to DNA damage involves regulation of multiple essential processes to maximize the accuracy of DNA damage repair and cell survival 1. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage, to increase the accuracy of repair. Here we report that telomerase action is regulated as a part of the cellular response to a DNA double-strand break (DSB). Using yeast, we show that the major ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. Upon DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Utilizing a separation of function PIF1 mutation, we show that this phosphorylation is required for the Pif1-mediated telomerase inhibition that takes place specifically at DNA breaks, but not telomeres. Hence DNA damage signalling down-modulates telomerase action at a DNA break via Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a novel regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity. PMID:19838171

  10. 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.

  11. Cellular responses to environmental DNA damage

    SciTech Connect

    Not Available

    1994-08-01

    This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

  12. A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints.

    PubMed

    Mu, Jung-Jung; Wang, Yi; Luo, Hao; Leng, Mei; Zhang, Jinglan; Yang, Tao; Besusso, Dario; Jung, Sung Yun; Qin, Jun

    2007-06-15

    ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

  13. DNA Damage in Plant Herbarium Tissue

    PubMed Central

    Staats, Martijn; Cuenca, Argelia; Richardson, James E.; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T.

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4–3.8% of fresh control DNA and 1.0–1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens. PMID:22163018

  14. DNA damage in major psychiatric diseases

    PubMed Central

    Raza, Muhammad Ummear; Tufan, Turan; Wang, Yan; Hill, Christopher; Zhu, Meng-Yang

    2016-01-01

    Human cells are exposed to exogenous insults and continuous production of different metabolites. These insults and unwanted metabolic products might interfere with the stability of genomic DNA. Recently many studies demonstrated that different psychiatric disorders show substantially high level of oxidative DNA damage in the brain accompanied with morphological and functional alterations. It reveals that damaged genomic DNA may contribute to the pathophysiology of these mental illnesses. Here we review the role of oxidative damage and reduced antioxidant ability in some vastly studied psychiatric disorders and emphasize the inclusion of treatment options involving DNA repair. In addition, while most currently used antidepressants are based on the manipulation of the neurotransmitter regulation in managing different mental abnormalities, they are able to prevent or reverse neurotoxin-induced DNA damage. Therefore, it may be plausible to target on genomic DNA alterations for psychiatric therapies, which is of pivotal importance for future anti-psychiatric drug development. PMID:27126805

  15. Dyskeratosis congenita and the DNA damage response

    PubMed Central

    Kirwan, Michael; Beswick, Richard; Walne, Amanda J; Hossain, Upal; Casimir, Colin; Vulliamy, Tom; Dokal, Inderjeet

    2011-01-01

    Dyskeratosis congenita (DC) is a heterogeneous bone marrow failure disorder with known mutations in components of telomerase and telomere shelterin. Recent work in a mouse model with a dyskerin mutation has implicated an increased DNA damage response as part of the cellular pathology, while mouse models with Terc and Tert mutations displayed a normal response. To clarify how these contradictory results might apply to DC pathology in humans, we studied the cellular phenotype in primary cells from DC patients of several genetic subtypes, focussing on T lymphocytes to remain close to the haematopoietic system. We observed novel cell cycle abnormalities in conjunction with impaired growth and an increase in apoptosis. Using flow cytometry and confocal microscopy we examined induction of the DNA damage proteins γ-H2AX and 53BP1 and the cell cycle protein TP53 (p53). We found an increase in damage foci at telomeres in lymphocytes and an increase in the basal level of DNA damage in fibroblasts, but crucially no increased response to DNA damaging agents in either cell type. As the response to induced DNA damage was normal and levels of global DNA damage were inconsistent between cell types, DNA damage may contribute differently to the pathology in different tissues. PMID:21477209

  16. Apoptosis and DNA damage in human spermatozoa

    PubMed Central

    Aitken, R John; Koppers, Adam J

    2011-01-01

    DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy. PMID:20802502

  17. 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.

  18. Surviving the breakup: the DNA damage checkpoint.

    PubMed

    Harrison, Jacob C; Haber, James E

    2006-01-01

    In response to even a single chromosomal double-strand DNA break, cells enact the DNA damage checkpoint. This checkpoint triggers cell cycle arrest, providing time for the cell to repair damaged chromosomes before entering mitosis. This mechanism helps prevent the segregation of damaged or mutated chromosomes and thus promotes genomic stability. Recent work has elucidated the molecular mechanisms underlying several critical steps in checkpoint activation, notably the recruitment of the upstream checkpoint kinases of the ATM and ATR families to different damaged DNA structures and the molecular events through which these kinases activate their effectors. Chromatin modification has emerged as one important component of checkpoint activation and maintenance. Following DNA repair, the checkpoint pathway is inactivated in a process termed recovery. A related but genetically distinct process, adaptation, controls cell cycle re-entry in the face of unrepairable damage.

  19. Mechanisms of DNA damage, repair, and mutagenesis.

    PubMed

    Chatterjee, Nimrat; Walker, Graham C

    2017-06-01

    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. Environ. Mol. Mutagen. 58:235-263, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  20. Ubiquitylation, neddylation and the DNA damage response

    PubMed Central

    Brown, Jessica S.; Jackson, Stephen P.

    2015-01-01

    Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair. PMID:25833379

  1. Ubiquitylation, neddylation and the DNA damage response.

    PubMed

    Brown, Jessica S; Jackson, Stephen P

    2015-04-01

    Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair.

  2. Triplex-induced DNA damage response.

    PubMed

    Rogers, Faye A; Tiwari, Meetu Kaushik

    2013-12-13

    Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage.

  3. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  4. Immunochemical detection of glyoxal DNA damage.

    PubMed

    Mistry, N; Evans, M D; Griffiths, H R; Kasai, H; Herbert, K E; Lunec, J

    1999-05-01

    The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.

  5. 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.

  6. 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

  7. Aging: not all DNA damage is equal.

    PubMed

    Vermeij, Wilbert P; Hoeijmakers, Jan H J; Pothof, Joris

    2014-06-01

    Recent advances have identified accumulation of DNA damage as a major driver of aging. However, there are numerous kinds of DNA lesions each with their own characteristics and cellular outcome, which highly depends on cellular context: proliferation (cell cycle), differentiation, propensity for survival/death, cell condition and systemic hormonal and immunological parameters. In addition, DNA damage is strongly influenced by cellular metabolism, anti-oxidant status and exogenous factors, consistent with the multi-factorial nature of aging. Notably, DNA lesions interfering with replication have very different outcomes compared to transcription. These considerations provide a conceptual framework in which different types of DNA damage and their setting contribute to the aging process in differential manners.

  8. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  9. Historical Perspective on the DNA Damage Response

    PubMed Central

    Hanawalt, Philip C.

    2015-01-01

    The DNA damage response (DDR) has been broadly defined as a complex network of cellular pathways that cooperate to sense and repair lesions in DNA. Multiple types of DNA damage, some natural DNA sequences, nucleotide pool deficiencies and collisions with transcription complexes can cause replication arrest to elicit the DDR. However, in practice, the term DDR as applied to eukaryotic/mammalian cells often refers more specifically to pathways involving the activation of the ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related) kinases in response to double-strand breaks or arrested replication forks, respectively. Nevertheless, there are distinct responses to particular types of DNA damage that do not involve ATM or ATR. In addition, some of the aberrations that cause replication arrest and elicit the DDR cannot be categorized as direct DNA damage. These include nucleotide pool deficiencies, nucleotide sequences that can adopt non-canonical DNA structures, and collisions between replication forks and transcription complexes. The response to these aberrations can be called the genomic stress response (GSR), a term that is meant to encompass the sensing of all types of DNA aberrations together with the mechanisms involved in coping with them. In addition to fully functional cells, the consequences of processing genomic aberrations may include mutagenesis, genomic rearrangements and lethality. PMID:26507443

  10. Historical perspective on the DNA damage response.

    PubMed

    Hanawalt, Philip C

    2015-12-01

    The DNA damage response (DDR) has been broadly defined as a complex network of cellular pathways that cooperate to sense and repair lesions in DNA. Multiple types of DNA damage, some natural DNA sequences, nucleotide pool deficiencies and collisions with transcription complexes can cause replication arrest to elicit the DDR. However, in practice, the term DDR as applied to eukaryotic/mammalian cells often refers more specifically to pathways involving the activation of the ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related) kinases in response to double-strand breaks or arrested replication forks, respectively. Nevertheless, there are distinct responses to particular types of DNA damage that do not involve ATM or ATR. In addition, some of the aberrations that cause replication arrest and elicit the DDR cannot be categorized as direct DNA damage. These include nucleotide pool deficiencies, nucleotide sequences that can adopt non-canonical DNA structures, and collisions between replication forks and transcription complexes. The response to these aberrations can be called the genomic stress response (GSR), a term that is meant to encompass the sensing of all types of DNA aberrations together with the mechanisms involved in coping with them. In addition to fully functional cells, the consequences of processing genomic aberrations may include mutagenesis, genomic rearrangements and lethality.

  11. DNA damage may drive nucleosomal reorganization to facilitate damage detection

    NASA Astrophysics Data System (ADS)

    LeGresley, Sarah E.; Wilt, Jamie; Antonik, Matthew

    2014-03-01

    One issue in genome maintenance is how DNA repair proteins find lesions at rates that seem to exceed diffusion-limited search rates. We propose a phenomenon where DNA damage induces nucleosomal rearrangements which move lesions to potential rendezvous points in the chromatin structure. These rendezvous points are the dyad and the linker DNA between histones, positions in the chromatin which are more likely to be accessible by repair proteins engaged in a random search. The feasibility of this mechanism is tested by considering the statistical mechanics of DNA containing a single lesion wrapped onto the nucleosome. We consider lesions which make the DNA either more flexible or more rigid by modeling the lesion as either a decrease or an increase in the bending energy. We include this energy in a partition function model of nucleosome breathing. Our results indicate that the steady state for a breathing nucleosome will most likely position the lesion at the dyad or in the linker, depending on the energy of the lesion. A role for DNA binding proteins and chromatin remodelers is suggested based on their ability to alter the mechanical properties of the DNA and DNA-histone binding, respectively. We speculate that these positions around the nucleosome potentially serve as rendezvous points where DNA lesions may be encountered by repair proteins which may be sterically hindered from searching the rest of the nucleosomal DNA. The strength of the repositioning is strongly dependent on the structural details of the DNA lesion and the wrapping and breathing of the nucleosome. A more sophisticated evaluation of this proposed mechanism will require detailed information about breathing dynamics, the structure of partially wrapped nucleosomes, and the structural properties of damaged DNA.

  12. DNA DAMAGE BINDING PROTEIN2 Shapes the DNA Methylation Landscape

    PubMed Central

    Schalk, Catherine; Kramdi, Amira; Ahmed, Ikhlak; Cognat, Valérie; Graindorge, Stéfanie; Bergdoll, Marc; Baumberger, Nicolas; Heintz, Dimitri; Bowler, Chris; Genschik, Pascal; Barneche, Fredy; Molinier, Jean

    2016-01-01

    In eukaryotes, DNA repair pathways help to maintain genome integrity and epigenomic patterns. However, the factors at the nexus of DNA repair and chromatin modification/remodeling remain poorly characterized. Here, we uncover a previously unrecognized interplay between the DNA repair factor DNA DAMAGE BINDING PROTEIN2 (DDB2) and the DNA methylation machinery in Arabidopsis thaliana. Loss-of-function mutation in DDB2 leads to genome-wide DNA methylation alterations. Genetic and biochemical evidence indicate that at many repeat loci, DDB2 influences de novo DNA methylation by interacting with ARGONAUTE4 and by controlling the local abundance of 24-nucleotide short interfering RNAs (siRNAs). We also show that DDB2 regulates active DNA demethylation mediated by REPRESSOR OF SILENCING1 and DEMETER LIKE3. Together, these findings reveal a role for the DNA repair factor DDB2 in shaping the Arabidopsis DNA methylation landscape in the absence of applied genotoxic stress. PMID:27531226

  13. Aging processes, DNA damage, and repair.

    PubMed

    Gilchrest, B A; Bohr, V A

    1997-04-01

    The second triennial FASEB Summer Research Conference on "Clonal Senescence and Differentiation" (August 17-22, 1996) focused on the interrelationships between aging processes and DNA damage and repair. The attendees represented a cross section of senior and junior investigators working in fields ranging from classic cellular gerontology to yeast and nematode models of aging to basic mechanisms of DNA damage and repair. The meeting opened with a keynote address by Dr. Bruce Ames that emphasized the documented relationships between oxidative damage, cancer, and aging. This was followed by eight platform sessions, one poster discussion, one featured presentation, and an after-dinner address. The following sections highlight the key points discussed.

  14. Polyphenols and DNA Damage: A Mixed Blessing

    PubMed Central

    Azqueta, Amaya; Collins, Andrew

    2016-01-01

    Polyphenols are a very broad group of chemicals, widely distributed in plant foods, and endowed with antioxidant activity by virtue of their numerous phenol groups. They are widely studied as putative cancer-protective agents, potentially contributing to the cancer preventive properties of fruits and vegetables. We review recent publications relating to human trials, animal experiments and cell culture, grouping them according to whether polyphenols are investigated in whole foods and drinks, in plant extracts, or as individual compounds. A variety of assays are in use to study genetic damage endpoints. Human trials, of which there are rather few, tend to show decreases in endogenous DNA damage and protection against DNA damage induced ex vivo in blood cells. Most animal experiments have investigated the effects of polyphenols (often at high doses) in combination with known DNA-damaging agents, and generally they show protection. High concentrations can themselves induce DNA damage, as demonstrated in numerous cell culture experiments; low concentrations, on the other hand, tend to decrease DNA damage. PMID:27918471

  15. Polyphenols and DNA Damage: A Mixed Blessing.

    PubMed

    Azqueta, Amaya; Collins, Andrew

    2016-12-03

    Polyphenols are a very broad group of chemicals, widely distributed in plant foods, and endowed with antioxidant activity by virtue of their numerous phenol groups. They are widely studied as putative cancer-protective agents, potentially contributing to the cancer preventive properties of fruits and vegetables. We review recent publications relating to human trials, animal experiments and cell culture, grouping them according to whether polyphenols are investigated in whole foods and drinks, in plant extracts, or as individual compounds. A variety of assays are in use to study genetic damage endpoints. Human trials, of which there are rather few, tend to show decreases in endogenous DNA damage and protection against DNA damage induced ex vivo in blood cells. Most animal experiments have investigated the effects of polyphenols (often at high doses) in combination with known DNA-damaging agents, and generally they show protection. High concentrations can themselves induce DNA damage, as demonstrated in numerous cell culture experiments; low concentrations, on the other hand, tend to decrease DNA damage.

  16. Targeting the DNA Damage Response in Cancer.

    PubMed

    O'Connor, Mark J

    2015-11-19

    An underlying hallmark of cancers is their genomic instability, which is associated with a greater propensity to accumulate DNA damage. Historical treatment of cancer by radiotherapy and DNA-damaging chemotherapy is based on this principle, yet it is accompanied by significant collateral damage to normal tissue and unwanted side effects. Targeted therapy based on inhibiting the DNA damage response (DDR) in cancers offers the potential for a greater therapeutic window by tailoring treatment to patients with tumors lacking specific DDR functions. The recent approval of olaparib (Lynparza), the poly (ADP-ribose) polymerase (PARP) inhibitor for treating tumors harboring BRCA1 or BRCA2 mutations, represents the first medicine based on this principle, exploiting an underlying cause of tumor formation that also represents an Achilles' heel. This review highlights the different concepts behind targeting DDR in cancer and how this can provide significant opportunities for DDR-based therapies in the future.

  17. Fern spore extracts can damage DNA

    PubMed Central

    Simán, S E; Povey, A C; Ward, T H; Margison, G P; Sheffield, E

    2000-01-01

    The carcinogenicity of the vegetative tissues of bracken fern (Pteridium) has long been established. More recently, the carcinogenic effects of the spores of bracken have also been recognized. Both vegetative tissues and spores of bracken can induce adducts in DNA in animal tissues, but the possible genotoxic or carcinogenic effects of spores from fern species other than bracken are unknown. The single-cell gel electrophoresis (‘comet’) assay was used to investigate whether fern spores can cause DNA damage in vitro. Extracts of spores from six fern species were administered to cultured human premyeloid leukaemia (K562) cells. Spore extracts of five fern species: Anemia phyllitidis, Dicksonia antarctica, Pteridium aquilinum, Pteris vittata and Sadleria pallida, induced significantly more DNA strand breaks than those in the control groups. Only in one species, Osmunda regalis, was the effect no different from that in the control groups. Using extracts from A. phyllitidis and P. vittata, the extent of DNA damage was increased by increasing the original dose 10 times, whereas an experiment in which exposure times were varied suggested that the highest levels of strand breaks appear after 2 h exposure. Simultaneous incubation with human S9 liver enzyme mix ablated the damaging effect of the extracts. Our data show that fern spore extracts can cause DNA damage in human cells in vitro. Considering the strong correlation between DNA damage and carcinogenic events, the observations made in this report may well have some implications for human health. © 2000 Cancer Research Campaign PMID:10883670

  18. 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.

  19. DNA Damages as a Depolymerization Process

    NASA Astrophysics Data System (ADS)

    Ochoa, Juan G. Diaz; Wulkow, Michael

    The damage of DNA chains by environmental factors like radiation or chemical pollutants is a topic that has been frequently explored from an experimental and a theoretical perspective. Such damages, like the damage of the strands of a DNA chain, are toxic for the cell and can induce mutagenesis or apoptosis. Several models make strong assumptions for the distribution of damages; for instance a frequent supposition is that these damages are Poisson distributed. [L. Ma, J. J. Wagner, W. Hu, A. J. Levine and G. A. Stolovitzki, Proc. Natl. Acad. Sci.PNAS 102, 14266 (2005).] Only few models describe in detail the damage and the mechanisms associated to the formation and evolution of this damage distribution [H. Nikjoo, P. O'neill and D. T. Goodhead, Radiat. Res. 156, 577 (2001).] Nevertheless, such models do not include the repair processes which are continuously active inside the cell. In this work we present a novel model, based on a depolymerization process, describing the distribution of damages on DNA chains coupled to the dynamics associated to its repair processes. The central aim is not to give a final and comprehensive model, but a hint to represent in more detail the complex dynamics involved in the damage and repair of DNA. We show that there are critical parameters associated to this repair process, in particular we show how critical doses can be relevant in deciding whether the cell continues its repair process or starts apoptosis. We also find out that the damage concentration is related to the dose via a power law relation.

  20. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  1. DNA damage, oxidative mutagen sensitivity, and repair of oxidative DNA damage in nonmelanoma skin cancer patients.

    PubMed

    Bendesky, Andrés; Michel, Alejandra; Sordo, Monserrat; Calderón-Aranda, Emma S; Acosta-Saavedra, Leonor C; Salazar, Ana M; Podoswa, Nancy; Ostrosky-Wegman, Patricia

    2006-08-01

    Nonmelanoma skin cancer (NMSC) is the most frequent type of cancer in humans. Exposure to UV radiation is a major risk factor for NMSC, and oxidative DNA damage, caused either by UV radiation itself or by other agents, may be involved in its induction. Increased sensitivity to oxidative damage and an altered DNA repair capacity (DRC) increase the risk of many types of cancer; however, sensitivity to oxidizing agents has not been evaluated for NMSC, and results regarding DRC in NMSC are inconclusive. In the present study, we evaluated DNA damage and repair in leukocytes from 41 NMSC patients and 45 controls. The Comet assay was used to measure basal and H(2)O(2)-induced DNA damage, as well as the DRC, while the cytokinesis-block micronucleus assay was used to measure the basal level of chromosome damage. Although basal DNA damage was higher for the controls than for the patients, this finding was mainly due to sampling more controls in the summer, which was associated with longer comet tails. In contrast, H(2)O(2)-induced DNA damage was significantly higher in cases than in controls, and this parameter was not influenced by the season of the year. The DRC for the H(2)O(2)-induced damage was similar for cases and controls and unrelated to seasonality. Finally, the frequency of binucleated lymphocytes with micronuclei was similar for cases and controls. The results of this study indicate that NMSC patients are distinguished from controls by an increased sensitivity to oxidative DNA damage.

  2. Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress

    PubMed Central

    Dong, Daoyin; Yu, Jingwen; Wu, Yanqing; Fu, Noah; Villela, Natalia Arias; Yang, Peixin

    2015-01-01

    DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy. PMID:26427872

  3. FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

    PubMed

    Castella, Maria; Jacquemont, Celine; Thompson, Elizabeth L; Yeo, Jung Eun; Cheung, Ronald S; Huang, Jen-Wei; Sobeck, Alexandra; Hendrickson, Eric A; Taniguchi, Toshiyasu

    2015-10-01

    The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

  4. FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2

    PubMed Central

    Castella, Maria; Jacquemont, Celine; Thompson, Elizabeth L.; Yeo, Jung Eun; Cheung, Ronald S.; Huang, Jen-Wei; Sobeck, Alexandra; Hendrickson, Eric A.; Taniguchi, Toshiyasu

    2015-01-01

    The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway. PMID:26430909

  5. Molecular Models for DNA Damaged by Photoreaction

    NASA Astrophysics Data System (ADS)

    Pearlman, David A.; Holbrook, Stephen R.; Pirkle, David H.; Kim, Sung-Hou

    1985-03-01

    Structural models of a DNA molecule containing a radiation-induced psoralen cross-link and of a DNA containing a thymine photodimer were constructed by applying energy-minimization techniques and model-building procedures to data from x-ray crystallographic studies. The helical axes of the models show substantial kinking and unwinding at the sites of the damage, which may have long-range as well as local effects arising from the concomitant changes in the supercoiling and overall structure of the DNA. The damaged areas may also serve as recognition sites for repair enzymes. These results should help in understanding the biologic effects of radiation-induced damage on cells.

  6. Molecular models for DNA damaged by photoreaction

    SciTech Connect

    Pearlman, D.A.; Holbrook, S.R.; Pirkle, D.H.; Kim, S.H.

    1985-03-15

    Structural models of a DNA molecule containing a radiation-induced psoralen cross-link and of a DNA containing a thymine photodimer were constructed by applying energy-minimization techniques and model-building procedures to data from x-ray crystallographic studies. The helical axes of the models show substantial kinking and unwinding at the sites of the damage, which may have long-range as well as local effects arising from the concomitant changes in the supercoiling and overall structure of the DNA. The damaged areas may also serve as recognition sites for repair enzymes. These results should help in understanding the biologic effects of radiation-induced damage on cells.

  7. Epigenome Maintenance in Response to DNA Damage.

    PubMed

    Dabin, Juliette; Fortuny, Anna; Polo, Sophie E

    2016-06-02

    Organism viability relies on the stable maintenance of specific chromatin landscapes, established during development, that shape cell functions and identities by driving distinct gene expression programs. Yet epigenome maintenance is challenged during transcription, replication, and repair of DNA damage, all of which elicit dynamic changes in chromatin organization. Here, we review recent advances that have shed light on the specialized mechanisms contributing to the restoration of epigenome structure and function after DNA damage in the mammalian cell nucleus. By drawing a parallel with epigenome maintenance during replication, we explore emerging concepts and highlight open issues in this rapidly growing field. In particular, we present our current knowledge of molecular players that support the coordinated maintenance of genome and epigenome integrity in response to DNA damage, and we highlight how nuclear organization impacts genome stability. Finally, we discuss possible functional implications of epigenome plasticity in response to genotoxic stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Epigenome maintenance in response to DNA damage

    PubMed Central

    Dabin, Juliette; Fortuny, Anna; Polo, Sophie E.

    2017-01-01

    Summary Organism viability relies on the stable maintenance of specific chromatin landscapes, established during development, that shape cell functions and identities by driving distinct gene expression programs. Yet, epigenome maintenance is challenged during transcription, replication and repair of DNA damage, all of which elicit dynamic changes in chromatin organization. Here, we review recent advances that have shed light on the specialized mechanisms contributing to the restoration of epigenome structure and function after DNA damage in the mammalian cell nucleus. By drawing a parallel with epigenome maintenance during replication, we explore emerging concepts and highlight open issues in this rapidly growing field. In particular, we present our current knowledge of molecular players that support the coordinated maintenance of genome and epigenome integrity in response to DNA damage, and we highlight how nuclear organization impacts genome stability. Finally, we discuss possible functional implications of epigenome plasticity in response to genotoxic stress. PMID:27259203

  9. Molecular mechanisms involved in initiation of the DNA damage response

    PubMed Central

    Barnum, Kevin J; O’Connell, Matthew J

    2015-01-01

    DNA is subject to a wide variety of damage. In order to maintain genomic integrity, cells must respond to this damage by activating repair and cell cycle checkpoint pathways. The initiating events in the DNA damage response entail recognition of the lesion and the assembly of DNA damage response complexes at the DNA. Here, we review what is known about these processes for various DNA damage pathways. PMID:27308403

  10. Molecular mechanisms involved in initiation of the DNA damage response.

    PubMed

    Barnum, Kevin J; O'Connell, Matthew J

    2015-01-01

    DNA is subject to a wide variety of damage. In order to maintain genomic integrity, cells must respond to this damage by activating repair and cell cycle checkpoint pathways. The initiating events in the DNA damage response entail recognition of the lesion and the assembly of DNA damage response complexes at the DNA. Here, we review what is known about these processes for various DNA damage pathways.

  11. The RNA Response to DNA Damage.

    PubMed

    Giono, Luciana E; Nieto Moreno, Nicolás; Cambindo Botto, Adrián E; Dujardin, Gwendal; Muñoz, Manuel J; Kornblihtt, Alberto R

    2016-06-19

    Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields.

  12. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    EPA Science Inventory

    This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

  13. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    EPA Science Inventory

    This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

  14. Alternative DNA Damage Checkpoint Pathways in Eukaryotes

    DTIC Science & Technology

    2000-04-01

    checkpoint pathway in Saccharomyces cerevisiae. Our hypothesis is that CHES1 does so by activating an alternative DNA damage-induced checkpoint pathway. The...difficulties, therefore we also tried the candidate gene and the yeast 2-hybrid approaches but with no success. In this report, we proposed alternative

  15. Profiling DNA damage response following mitotic perturbations

    PubMed Central

    S. Pedersen, Ronni; Karemore, Gopal; Gudjonsson, Thorkell; Rask, Maj-Britt; Neumann, Beate; Hériché, Jean-Karim; Pepperkok, Rainer; Ellenberg, Jan; Gerlich, Daniel W.; Lukas, Jiri; Lukas, Claudia

    2016-01-01

    Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes. PMID:27976684

  16. Immunochemical detection of oxidatively damaged DNA.

    PubMed

    Rossner, Pavel; Sram, Radim J

    2012-04-01

    Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, (32)P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.

  17. Oxidative and alkylating damage in DNA.

    PubMed

    Martinez, Glaucia R; Loureiro, Ana Paula M; Marques, Sabrina A; Miyamoto, Sayuri; Yamaguchi, Lydia F; Onuki, Janice; Almeida, Eduardo A; Garcia, Camila C M; Barbosa, Lívea F; Medeiros, Marisa H G; Di Mascio, Paolo

    2003-11-01

    Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

  18. Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress.

    PubMed

    Dong, Daoyin; Yu, Jingwen; Wu, Yanqing; Fu, Noah; Villela, Natalia Arias; Yang, Peixin

    2015-11-13

    DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy.

  19. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs.

    PubMed

    Rossiello, Francesca; Aguado, Julio; Sepe, Sara; Iannelli, Fabio; Nguyen, Quan; Pitchiaya, Sethuramasundaram; Carninci, Piero; d'Adda di Fagagna, Fabrizio

    2017-02-27

    The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.

  20. DNA damage checkpoint recovery and cancer development

    SciTech Connect

    Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong; Legerski, Randy J.

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  1. Telomeres, histone code, and DNA damage response.

    PubMed

    Misri, S; Pandita, S; Kumar, R; Pandita, T K

    2008-01-01

    Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability.

  2. Telomeres, histone code, and DNA damage response

    PubMed Central

    Misri, S.; Pandita, S.; Kumar, R.; Pandita, T.K.

    2009-01-01

    Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability. PMID:19188699

  3. DNA mismatch repair and the DNA damage response

    PubMed Central

    Li, Zhongdao; Pearlman, Alexander H.; Hsieh, Peggy

    2015-01-01

    This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted. PMID:26704428

  4. DNA mismatch repair and the DNA damage response.

    PubMed

    Li, Zhongdao; Pearlman, Alexander H; Hsieh, Peggy

    2016-02-01

    This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted. Published by Elsevier B.V.

  5. Immunofluorescence Imaging of DNA Damage Response Proteins

    PubMed Central

    Bennett, Brian T.; Bewersdorf, Jörg; Knight, Kendall L.

    2013-01-01

    Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage. PMID:19245833

  6. Dynamical signature of abasic damage in DNA.

    PubMed

    Furse, Kristina E; Corcelli, Steven A

    2011-02-02

    Time-dependent Stokes shift (TDSS) responses in proteins and DNA exhibit a broad range of long time scales (>10 ps) that are not present in bulk aqueous solution. The physical interpretation of the long TDSS time scales in biomolecular systems is a matter of considerable debate because of the many different components present in the sample (water, biomolecule, counterions), which have highly correlated motions and intrinsically different abilities to adapt to local perturbations. Here we use molecular dynamics (MD) simulations to show that the surprisingly slow (∼10 ns) TDSS response of coumarin 102 (C102), a base pair replacement, reflects a distinct dynamical signature for DNA damage. When the C102 molecule is covalently incorporated into DNA, an abasic site is created on the strand opposite the C102 probe. The abasic sugar exhibits a reversible interchange between intra- and extrahelical conformations that are kinetically stable on a nanosecond time scale. This conformational change, only possible in damaged DNA, was found to be responsible for the long time scales in the measured TDSS response. For the first time, a TDSS measurement has been attributed to a specific biomolecular motion. This finding directly contradicts the prevailing notion that the TDSS response in biomolecular contexts is dominated by hydration dynamics. It also suggests that TDSS experiments can be used to study ultrafast biomolecular dynamics that are inaccessible to other techniques.

  7. Profiling oxidative DNA damage: effects of antioxidants.

    PubMed

    Box, Harold C; Patrzyc, Helen B; Budzinski, Edwin E; Dawidzik, Jean B; Freund, Harold G; Zeitouni, Nathalie C; Mahoney, Martin C

    2012-11-01

    The goal of this research was to determine whether antioxidant usage could be correlated with changes in DNA damage levels. Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) was used to simultaneously measure five different oxidatively-induced base modifications in the DNA of WBC. Measurements of the five modifications were made before and after an 8-week trial during which participants took the SU.VI.MAX supplement. Levels of the five DNA modifications were compared among different groupings: users versus non-users of antioxidant supplements, before versus after the supplement intervention and men versus women. The statistical significance of differences between groups was most significant for pyrimidine base modifications and the observed trends reflect trends reported in epidemiological studies of antioxidant usage. A combination of modifications derived from pyrimidine bases is suggested as a superior indicator of oxidative stress.

  8. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  9. DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1.

    PubMed

    Selvarajah, Jogitha; Elia, Androulla; Carroll, Veronica A; Moumen, Abdeladim

    2015-01-01

    mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.

  10. DNA damage and innate immunity: links and trade-offs.

    PubMed

    Chatzinikolaou, Georgia; Karakasilioti, Ismene; Garinis, George A

    2014-09-01

    To counteract DNA damage, cells employ genome maintenance pathways that are directed inward, relentlessly to scan and repair the genome. Adaptive and innate immune mechanisms are often directed outward, protecting self against pathogens. Recent work has revealed direct links between innate immune signaling and the DNA damage response (DDR). Here we review current understanding of the mechanism by which cells sense damaged and foreign DNA. We examine the functional role of DNA damage signaling in immune activation and discuss the relevance of these processes to DNA damage-driven chronic inflammation in disease and in aging. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Acrylonitrile-induced oxidative DNA damage in rat astrocytes.

    PubMed

    Pu, Xinzhu; Kamendulis, Lisa M; Klaunig, James E

    2006-10-01

    Chronic administration of acrylonitrile results in a dose-related increase in astrocytomas in rat brain, but the mechanism of acrylonitrile carcinogenicity is not fully understood. The potential of acrylonitrile or its metabolites to induce direct DNA damage as a mechanism for acrylonitrile carcinogenicity has been questioned, and recent studies indicate that the mechanism involves the induction of oxidative stress in rat brain. The present study examined the ability of acrylonitrile to induce DNA damage in the DI TNC1 rat astrocyte cell line using the alkaline Comet assay. Oxidized DNA damage also was evaluated using formamidopyrimidine DNA glycosylase treatment in the modified Comet assay. No increase in direct DNA damage was seen in astrocytes exposed to sublethal concentrations of acrylonitrile (0-1.0 mM) for 24 hr. However, acrylonitrile treatment resulted in a concentration-related increase in oxidative DNA damage after 24 hr. Antioxidant supplementation in the culture media (alpha-tocopherol, (-)-epigallocathechin-3 gallate, or trolox) reduced acrylonitrile-induced oxidative DNA damage. Depletion of glutathione using 0.1 mM DL-buthionine-[S,R]-sulfoximine increased acrylonitrile-induced oxidative DNA damage (22-46%), while cotreatment of acrylonitrile with 2.5 mM L-2-oxothiazolidine-4-carboxylic acid, a precursor for glutathione biosynthesis, significantly reduced acrylonitrile-induced oxidative DNA damage (7-47%). Cotreatment of acrylonitrile with 0.5 mM 1-aminobenzotriazole, a suicidal inhibitor of cytochrome P450, prevented the oxidative DNA damage produced by acrylonitrile. Cyanide (0.1-0.5 mM) increased oxidative DNA damage (44-160%) in astrocytes. These studies demonstrate that while acrylonitrile does not directly damage astrocyte DNA, it does increase oxidative DNA damage. The oxidative DNA damage following acrylonitrile exposure appears to arise mainly through the P450 metabolic pathway; moreover, glutathione depletion may contribute to the

  12. Mitochondrial DNA damage induced autophagy, cell death, and disease.

    PubMed

    Van Houten, Bennett; Hunter, Senyene E; Meyer, Joel N

    2016-01-01

    Mammalian mitochondria contain multiple small genomes. While these organelles have efficient base excision removal of oxidative DNA lesions and alkylation damage, many DNA repair systems that work on nuclear DNA damage are not active in mitochondria. What is the fate of DNA damage in the mitochondria that cannot be repaired or that overwhelms the repair system? Some forms of mitochondrial DNA damage can apparently trigger mitochondrial DNA destruction, either via direct degradation or through specific forms of autophagy, such as mitophagy. However, accumulation of certain types of mitochondrial damage, in the absence of DNA ligase III (Lig3) or exonuclease G (EXOG), can directly trigger cell death. This review examines the cellular effects of persistent damage to mitochondrial genomes and discusses the very different cell fates that occur in response to different kinds of damage.

  13. Eukaryotic DNA damage responses: Homologous recombination factors and ubiquitin modification.

    PubMed

    Lee, Nam Soo; Kim, Soomi; Jung, Yong Woo; Kim, Hongtae

    2017-05-06

    To prevent genomic instability disorders, cells have developed a DNA damage response. The response involves various proteins that sense damaged DNA, transduce damage signals, and effect DNA repair. In addition, ubiquitin modifications modulate the signaling pathway depending on cellular context. Among various types of DNA damage, double-stranded breaks are highly toxic to genomic integrity. Homologous recombination (HR) repair is an essential mechanism that fixes DNA damage because of its high level of accuracy. Although factors in the repair pathway are well established, pinpointing the exact mechanisms of repair and devising therapeutic applications requires more studies. Moreover, essential functions of ubiquitin modification in the DNA damage signaling pathway have emerged. In this review, to explore the eukaryotic DNA damage response, we will mention the functions of main factors in the HR repair pathway and ubiquitin modification. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Salicylic Acid Activates DNA Damage Responses to Potentiate Plant Immunity

    PubMed Central

    Yan, Shunping; Wang, Wei; Marqués, Jorge; Mohan, Rajinikanth; Saleh, Abdelaty; Durrant, Wendy E.; Song, Junqi; Dong, Xinnian

    2013-01-01

    SUMMARY DNA damage is normally detrimental to living organisms. Here we show that it can also serve as a signal to promote immune responses in plants. We found that the plant immune hormone salicylic acid (SA) can trigger DNA damage in the absence of a genotoxic agent. The DNA damage sensor proteins, RAD17 and ATR, are required for effective immune responses. These sensor proteins are negatively regulated by a key immune regulator SNI1 (suppressor of npr1-1, inducible 1), which we discovered as a missing subunit of the Structural Maintenance of Chromosome (SMC) 5/6 complex required for controlling DNA damage. Elevated DNA damage caused by the sni1 mutation or treatment with a DNA-damaging agent markedly enhances SA-mediated defense gene expression. Our study suggests that activation of DNA damage responses is an intrinsic component of the plant immune responses. PMID:24207055

  15. Inflammation, oxidative DNA damage, and carcinogenesis.

    PubMed Central

    Lewis, J G; Adams, D O

    1987-01-01

    Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is thought that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. Many studies support the multistep nature of carcinogenesis, and a significant amount of evidence indicates that more than one genetic event is necessary for neoplastic transformation. Selective growth stimulation of initiated cells by TPA does not explain how further genetic events may occur by chronic exposure to this nongenotoxic agent. We and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H2O2 and oxidized lipid products. Furthermore, macrophage populations that release both H2O2 and metabolites of arachidonic acid (AA) are more efficient at inducing oxidative DNA damage in surrounding cells than populations which only release H2O2 or metabolites of AA. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin than C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H2O2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 8. A FIGURE 8. B PMID:3129286

  16. DNA-damaging agents from Crypteronia paniculata.

    PubMed

    Deng, Jing-Zhen; Marshall, Rebekah; Jones, Shannon H; Johnson, Randall K; Hecht, Sidney M

    2002-12-01

    A survey of crude plant extracts using a new yeast strain designed to identify DNA-damaging agents resulted in the identification of an extract prepared from Crypteronia paniculata. Bioassay-guided fractionation resulted in the isolation of three active compounds. Two of these were ellagic acid derivatives, namely, 3,3'-di-O-methylellagic acid 4'-O-beta-d-xylopyranoside (1) and 3'-O-methyl-3,4-methylenedioxyellagic acid 4'-O-beta-d-glucopyranoside (2). The third was identified as kaempferol-3-O-alpha-l-rhamnoside (3). The three principles exhibited strong, selective cytotoxity toward the RAD52 repair-deficient yeast strain.

  17. Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures.

    PubMed

    Sar, Funda; Lindsey-Boltz, Laura A; Subramanian, Deepa; Croteau, Deborah L; Hutsell, Stephanie Q; Griffith, Jack D; Sancar, Aziz

    2004-09-17

    Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.

  18. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

    PubMed Central

    Nelson, W G; Kastan, M B

    1994-01-01

    The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging

  19. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis.

    PubMed

    Roos, Wynand P; Kaina, Bernd

    2013-05-28

    DNA damaging agents are potent inducers of cell death triggered by apoptosis. Since these agents induce a plethora of different DNA lesions, it is firstly important to identify the specific lesions responsible for initiating apoptosis before the apoptotic executing pathways can be elucidated. Here, we describe specific DNA lesions that have been identified as apoptosis triggers, their repair and the signaling provoked by them. We discuss methylating agents such as temozolomide, ionizing radiation and cisplatin, all of them are important in cancer therapy. We show that the potentially lethal events for the cell are O(6)-methylguanine adducts that are converted by mismatch repair into DNA double-strand breaks (DSBs), non-repaired N-methylpurines and abasic sites as well as bulky adducts that block DNA replication leading to DSBs that are also directly induced following ionizing radiation. Transcriptional inhibition may also contribute to apoptosis. Cells are equipped with sensors that detect DNA damage and relay the signal via kinases to executors, who on their turn evoke a process that inhibits cell cycle progression and provokes DNA repair or, if this fails, activate the receptor and/or mitochondrial apoptotic cascade. The main DNA damage recognition factors MRN and the PI3 kinases ATM, ATR and DNA-PK, which phosphorylate a multitude of proteins and thus induce the DNA damage response (DDR), will be discussed as well as the downstream players p53, NF-κB, Akt and survivin. We review data and models describing the signaling from DNA damage to the apoptosis executing machinery and discuss the complex interplay between cell survival and death. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  20. DNA damage in normally and prematurely aged mice

    PubMed Central

    Maslov, Alexander Y.; Ganapathi, Shireen; Westerhof, Maaike; Quispe, Wilber; White, Ryan R.; Van Houten, Bennett; Reiling, Erwin; Dollé, Martijn E.T.; van Steeg, Harry; Hasty, Paul; Hoeijmakers, Jan H.J.; Vijg, Jan

    2013-01-01

    Summary Steady-state levels of spontaneous DNA damage, the by-product of normal metabolism and environmental exposure, are controlled by DNA repair pathways. Incomplete repair or an age-related increase in damage production and/or decline in repair could lead to an accumulation of DNA damage, increasing mutation rate, affecting transcription and/or activating programmed cell death or senescence. These consequences of DNA damage metabolism are highly conserved and the accumulation of lesions in the DNA of the genome could, therefore, provide a universal cause of aging. An important corollary of this hypothesis is that defects in DNA repair cause both premature aging and accelerated DNA damage accumulation. While the former has been well-documented, the reliable quantification of the various lesions thought to accumulate in DNA during aging has been a challenge. Here, we quantified inhibition of long distance PCR as a measure of DNA damage in liver and brain of both normal and prematurely aging, DNA repair defective mice. The results indicate a marginal, but statistically significant, increase of spontaneous DNA damage with age in normal mouse liver but not in brain. Increased levels of DNA damage were not observed in the DNA repair defective mice. We also show that oxidative lesions do not increase with age. These results indicate that neither normal nor premature aging is accompanied by a dramatic increase in DNA damage. This suggests that factors other than DNA damage per se, e.g., cellular responses to DNA damage, are responsible for the aging phenotype in mice. PMID:23496256

  1. DNA damage tolerance by recombination: Molecular pathways and DNA structures.

    PubMed

    Branzei, Dana; Szakal, Barnabas

    2016-08-01

    Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  2. DNA Damage Response and Immune Defense: Links and Mechanisms.

    PubMed

    Nakad, Rania; Schumacher, Björn

    2016-01-01

    DNA damage plays a causal role in numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR) orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signaling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signaling. We highlight evidence gained into (i) which molecular and cellular pathways of DDR activate immune signaling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans.

  3. DNA Damage Response and Immune Defense: Links and Mechanisms

    PubMed Central

    Nakad, Rania; Schumacher, Björn

    2016-01-01

    DNA damage plays a causal role in numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR) orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signaling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signaling. We highlight evidence gained into (i) which molecular and cellular pathways of DDR activate immune signaling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans. PMID:27555866

  4. Inflammation, oxidative DNA damage, and carcinogenesis

    SciTech Connect

    Lewis, J.G.; Adams, D.O.

    1987-12-01

    Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is though that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. The authors and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H/sub 2/O/sub 2/ and oxidized lipid products. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin that C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H/sub 2/O/sub 2/ and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice. These data support the hypothesis that inflammation and the release of genotoxic oxidants may be one mechanism whereby initiated cells receive further genetic insults. They also further complicate risk assessment by suggesting that some environmental agents may work indirectly by subverting host systems to induce damage rather than maintaining homeostasis.

  5. Replication fork dynamics and the DNA damage response.

    PubMed

    Jones, Rebecca M; Petermann, Eva

    2012-04-01

    Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

  6. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs

    PubMed Central

    Rossiello, Francesca; Aguado, Julio; Sepe, Sara; Iannelli, Fabio; Nguyen, Quan; Pitchiaya, Sethuramasundaram; Carninci, Piero; d’Adda di Fagagna, Fabrizio

    2017-01-01

    The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs. PMID:28239143

  7. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities.

    PubMed

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K

    2014-08-01

    Loss of Werner syndrome protein function causes Werner syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN's DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor HU. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency.

  8. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  9. Radiation damage to DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Spotheim-Maurizot, M.; Davídková, M.

    2011-01-01

    We review here the advances in understanding the effects of ionizing radiations on DNA, proteins and their complexes, resulting from the collaboration of the authors' teams. It concerns the preponderant indirect effect of low LET ionizing radiations, thus the attack of the macromolecules in aqueous solution by the most aggressive product of water radiolysis, the hydroxyl radical. A model of simulation of the reaction of these radicals with the macromolecules (called RADACK) was developed and was used for calculating the probabilities of damage of each constituent of DNA or proteins (nucleotide or amino-acid). The calculations allowed to draw conclusions from electrophoresis, mutagenesis, spectroscopic (fluorescence, circular dichroïsm) and mass spectrometry experiments. Thus we have shown that the extent and location of the lesions are strongly dependent on the 3D structure of the macromolecules, which in turns is modulated by their sequence and by the binding of some ligands. Molecular dynamics simulation completed our studies in showing the consequences of each lesion on the stability and structure of the proteins and their complexes with DNA.

  10. Platinum nanoparticles induce damage to DNA and inhibit DNA replication

    PubMed Central

    Nejdl, Lukas; Kudr, Jiri; Moulick, Amitava; Hegerova, Dagmar; Ruttkay-Nedecky, Branislav; Gumulec, Jaromir; Cihalova, Kristyna; Smerkova, Kristyna; Dostalova, Simona; Krizkova, Sona; Novotna, Marie; Kopel, Pavel

    2017-01-01

    Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8–11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent. PMID:28704436

  11. DNA damage in Fabry patients: An investigation of oxidative damage and repair.

    PubMed

    Biancini, Giovana Brondani; Moura, Dinara Jaqueline; Manini, Paula Regina; Faverzani, Jéssica Lamberty; Netto, Cristina Brinckmann Oliveira; Deon, Marion; Giugliani, Roberto; Saffi, Jenifer; Vargas, Carmen Regla

    2015-06-01

    Fabry disease (FD) is a lysosomal storage disorder associated with loss of activity of the enzyme α-galactosidase A. In addition to accumulation of α-galactosidase A substrates, other mechanisms may be involved in FD pathophysiology, such as inflammation and oxidative stress. Higher levels of oxidative damage to proteins and lipids in Fabry patients were previously reported. However, DNA damage by oxidative species in FD has not yet been studied. We investigated basal DNA damage, oxidative DNA damage, DNA repair capacity, and reactive species generation in Fabry patients and controls. To measure oxidative damage to purines and pyrimidines, the alkaline version of the comet assay was used with two endonucleases, formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (EndoIII). To evaluate DNA repair, a challenge assay with hydrogen peroxide was performed. Patients presented significantly higher levels of basal DNA damage and oxidative damage to purines. Oxidative DNA damage was induced in both DNA bases by H2O2 in patients. Fabry patients presented efficient DNA repair in both assays (with and without endonucleases) as well as significantly higher levels of oxidative species (measured by dichlorofluorescein content). Even if DNA repair be induced in Fabry patients (as a consequence of continuous exposure to oxidative species), the repair is not sufficient to reduce DNA damage to control levels.

  12. 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

  13. 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

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

    PubMed

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

    2014-06-01

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

  15. 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.

  16. Hydroxyl radical Thymine adduct induced DNA damages

    NASA Astrophysics Data System (ADS)

    Schyman, Patric; Eriksson, Leif A.; Zhang, Ru bo; Laaksonen, Aatto

    2008-06-01

    DNA damages caused by a 5-hydroxy-5,6-dihydrothymine-6-yl radical (5-OHT-6yl) abstracting a C2‧ hydrogen from a neighboring sugar (inter-H abstraction) have been theoretically investigated using hybrid DFT in gas phase and in water solution. The inter-H abstraction was here shown to be comparable in energy (24 kcal mol-1) with the intra-H abstraction in which the 5-OHT-6yl abstracts a C2‧ hydrogen from its own sugar. The effect of a neutrally or a negatively charged phosphate group was also studied and the results show no significant impact on the activation energy of the hydrogen abstraction whereas base release and strand break reactions are affected.

  17. RNF111-dependent neddylation activates DNA damage-induced ubiquitination

    PubMed Central

    Ma, Teng; Chen, Yibin; Zhang, Feng; Yang, Chao-Yie; Wang, Shaomeng; Yu, Xiaochun

    2013-01-01

    Summary Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites, and this accumulation was dependent on an E2 enzyme UBE2M and an E3 ubiquitin ligase RNF111. We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (Motif Interacting with Ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process. PMID:23394999

  18. Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.

    PubMed

    Marheineke, Kathrin; Hyrien, Olivier

    2004-07-02

    A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close ( approximately 10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase.

  19. Mitochondrial DNA damage by bleomycin induces AML cell death.

    PubMed

    Yeung, ManTek; Hurren, Rose; Nemr, Carine; Wang, Xiaoming; Hershenfeld, Samantha; Gronda, Marcela; Liyanage, Sanduni; Wu, Yan; Augustine, Jeevan; Lee, Eric A; Spagnuolo, Paul A; Southall, Noel; Chen, Catherine; Zheng, Wei; Jeyaraju, Danny V; Minden, Mark D; Laposa, Rebecca; Schimmer, Aaron D

    2015-06-01

    Mitochondria contain multiple copies of their own 16.6 kb circular genome. To explore the impact of mitochondrial DNA (mtDNA) damage on mitochondrial (mt) function and viability of AML cells, we screened a panel of DNA damaging chemotherapeutic agents to identify drugs that could damage mtDNA. We identified bleomycin as an agent that damaged mtDNA in AML cells at concentrations that induced cell death. Bleomycin also induced mtDNA damage in primary AML samples. Consistent with the observed mtDNA damage, bleomycin reduced mt mass and basal oxygen consumption in AML cells. We also demonstrated that the observed mtDNA damage was functionally important for bleomycin-induced cell death. Finally, bleomycin delayed tumor growth in xenograft mouse models of AML and anti-leukemic concentrations of the drug induced mtDNA damage in AML cells preferentially over normal lung tissue. Taken together, mtDNA-targeted therapy may be an effective strategy to target AML cells and bleomycin could be useful in the treatment of this disease.

  20. Commentary: Mitochondrial DNA damage and loss in diabetes

    PubMed Central

    Gilkerson, Robert

    2017-01-01

    This commentary discusses damage and loss of mitochondrial DNA (mtDNA) in type 2 diabetes mellitus from both the clinical and experimental perspectives. Increasingly, an array of studies in experimental models and patients suggests that the cellular stresses of insulin resistance in type 2 diabetes damage mtDNA, leading to loss of mitochondrial genetic content. As such, mtDNA is emerging as both a valuable monitoring tool and translational preventive target for metabolic disease. PMID:27253402

  1. Oxidative DNA damage during night shift work.

    PubMed

    Bhatti, Parveen; Mirick, Dana K; Randolph, Timothy W; Gong, Jicheng; Buchanan, Diana Taibi; Zhang, Junfeng Jim; Davis, Scott

    2017-09-01

    We previously reported that compared with night sleep, day sleep among shift workers was associated with reduced urinary excretion of 8-hydroxydeoxyguanosine (8-OH-dG), potentially reflecting a reduced ability to repair 8-OH-dG lesions in DNA. We identified the absence of melatonin during day sleep as the likely causative factor. We now investigate whether night work is also associated with reduced urinary excretion of 8-OH-dG. For this cross-sectional study, 50 shift workers with the largest negative differences in night work versus night sleep circulating melatonin levels (measured as 6-sulfatoxymelatonin in urine) were selected from among the 223 shift workers included in our previous study. 8-OH-dG concentrations were measured in stored urine samples using high performance liquid chromatography with electrochemical detection. Mixed effects models were used to compare night work versus night sleep 8-OH-dG levels. Circulating melatonin levels during night work (mean=17.1 ng/mg creatinine/mg creatinine) were much lower than during night sleep (mean=51.7 ng/mg creatinine). In adjusted analyses, average urinary 8-OH-dG levels during the night work period were only 20% of those observed during the night sleep period (95% CI 10% to 30%; p<0.001). This study suggests that night work, relative to night sleep, is associated with reduced repair of 8-OH-dG lesions in DNA and that the effect is likely driven by melatonin suppression occurring during night work relative to night sleep. If confirmed, future studies should evaluate melatonin supplementation as a means to restore oxidative DNA damage repair capacity among shift workers. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  2. Impact of Alternative DNA Structures on DNA Damage, DNA Repair, and Genetic Instability

    PubMed Central

    Wang, Guliang; Vasquez, Karen M.

    2014-01-01

    Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability. PMID:24767258

  3. Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924.

    PubMed

    Blank, Jonathan L; Liu, Xiaozhen J; Cosmopoulos, Katherine; Bouck, David C; Garcia, Khristofer; Bernard, Hugues; Tayber, Olga; Hather, Greg; Liu, Ray; Narayanan, Usha; Milhollen, Michael A; Lightcap, Eric S

    2013-01-01

    MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

  4. Saccharomyces cerevisiae-based system for studying clustered DNA damages

    SciTech Connect

    Moscariello, M.M.; Sutherland, B.

    2010-08-01

    DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents.

  5. Systemic DNA damage responses in aging and diseases

    PubMed Central

    Ribezzo, Flavia; Shiloh, Yosef; Schumacher, Björn

    2016-01-01

    The genome is constantly attacked by a variety of genotoxic insults. The causal role for DNA damage in aging and cancer is exemplified by genetic defects in DNA repair that underlie a broad spectrum of acute and chronic human disorders that are characterized by developmental abnormalities, premature aging, and cancer predisposition. The disease symptoms are typically tissue-specific with uncertain genotype-phenotype correlation. The cellular DNA damage response (DDR) has been extensively investigated ever since yeast geneticists discovered DNA damage checkpoint mechanisms, several decades ago. In recent years, it has become apparent that not only cell-autonomous but also systemic DNA damage responses determine the outcome of genome instability in organisms. Understanding the mechanisms of non-cell-autonomous DNA damage responses will provide important new insights into the role of genome instability in human aging and a host of diseases including cancer and might better explain the complex phenotypes caused by genome instability. PMID:26773346

  6. 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.

  7. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  8. Novel DNA damage checkpoint in mitosis: Mitotic DNA damage induces re-replication without cell division in various cancer cells.

    PubMed

    Hyun, Sun-Yi; Rosen, Eliot M; Jang, Young-Joo

    2012-07-06

    DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair.

  9. DNA damage induced by low energy electron collision and new experimental setup for further studying DNA damage by plasma

    NASA Astrophysics Data System (ADS)

    Park, Yeunsoo; Sanche, Leon; Wagner, Richard

    2013-09-01

    Low energy electrons (LEEs; below 10 eV) are the most abundant among the radiolytic species generated along the high energy radiation track in living cell. And these electrons are also one of major components with ions and photon in plasma. Interestingly, it has turned out that LEEs can create DNA damages such as base release, single- and double- strand breaks (SSB and DSB) via indirect action named dissociative electron attachment (DEA). The purposes of this study are to further find out exact mechanisms of DNA damage by LEEs at the molecular level and to verify new DNA damage like structural alteration on DNA subunits. And we will expand our study to DNA damage by plasma source to develop plasma-based new medical and biological applications. We are currently setting new experimental system for reaching our goals. We will show some recent results about new finding DNA modification damage and some experimental designs and working principles.

  10. Oxidative DNA damage in osteoarthritic porcine articular cartilage

    PubMed Central

    Chen, Antonia F.; Davies, Catrin M.; De Lin, Ming; Fermor, Beverley

    2008-01-01

    Purpose Osteoarthritis (OA) is associated with increased levels of reactive oxygen species. This study investigated if increased oxidative DNA damage accumulates in OA articular cartilage compared with non-OA articular cartilage from pigs with spontaneous OA. Additionally, the ability of nitric oxide (NO) or peroxynitrite (ONOO-) induced DNA damage in non-OA chondrocytes to undergo endogenous repair was investigated. Methods Porcine femoral condyles were graded for the stage of OA, macroscopically by the Collins Scale, and histologically by the modified Mankin Grade. Levels of DNA damage were determined in non-OA and OA cartilage, using the comet assay. For calibration, DNA damage was measured by exposing non-OA chondrocytes to 0-12 Gray of x-ray irradiation. Non-OA articular chondrocytes were treated with 0-500 μM of NO donors (NOC-18 or SIN-1), and DNA damage assessed after treatment and 5 days recovery. Results A significant increase (p<0.01) in oxidative DNA damage occurred in OA chondrocytes in joints with Mankin Grades 3 or greater, compared to non-OA chondrocytes. The percentage of nuclei containing DNA damage increased significantly (p<0.001) from early to late grades of OA. An increase of approximately 0.65-1.7 breaks/1000kB of DNA occurred in OA, compared to non-OA nuclei. NOC-18 or SIN-1 caused significant DNA damage (p<0.001) in non-OA chondrocytes that did not undergo full endogenous repair after 5 days (p<0.05). Conclusion Our data suggest significant levels of oxidative DNA damage occur in OA chondrocytes that accumulates with OA progression. Additionally, DNA damage induced by NO and ONOO- in non-OA chondrocytes does not undergo full endogenous repair. PMID:18720406

  11. DNA damage in cells exhibiting radiation-induced genomic instability

    SciTech Connect

    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 hypothesis 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.

  12. 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

  13. 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

  14. Guarding chromosomes from oxidative DNA damage to the very end.

    PubMed

    Tan, Rong; Lan, Li

    2016-07-01

    The ends of each chromosome are capped by the telomere assembly to protect chromosomal integrity from telomere attrition and DNA damage. In response to DNA damage, DNA repair factors are enriched at damage sites by a sophisticated signaling and recruitment cascade. However, DNA damage response at telomeres is different from non-telomeric region of genomic DNA due to specialized sequences and structures of the telomeres. In the course of normal DNA replication or DNA damage repair, both the telomere shelterin protein complex and the condensed telomeric chromatin structure in mammalian cells are modified to protect telomeres from exposing free DNA ends which are subject to both telemere shortening and chromosome end fusion. Initiation of either homologous recombination or non-homologous end joint repair at telomeres requires disassembling and/or post-translational modifications of the shelterin complex and telomeric chromatin. In addition, cancer cells utilize distinct mechanisms to maintain telomere length and cell survival upon damage. In this review, we summarize current studies that focus on telomere end protection and telomere DNA repair using different methodologies to model telomere DNA damage and disruption. These include genetic ablation of sheltering proteins, targeting endonuclease to telomeres, and delivering oxidative damage directly. These different approaches, when combined, offer better understanding of the mechanistic differences in DNA damage response between telomeric and genomic DNA, which will provide new hope to identify potential cancer therapeutic targets to curtail cancer cell proliferation via induction of telomere dysfunctions. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Inflammation, DNA Damage, Helicobacter pylori and Gastric Tumorigenesis

    PubMed Central

    Kalisperati, Polyxeni; Spanou, Evangelia; Pateras, Ioannis S.; Korkolopoulou, Penelope; Varvarigou, Anastasia; Karavokyros, Ioannis; Gorgoulis, Vassilis G.; Vlachoyiannopoulos, Panayiotis G.; Sougioultzis, Stavros

    2017-01-01

    Helicobacter pylori (H. pylori) is a Gram negative bacterium that colonizes the stomach of almost half human population. It has evolved to escape immune surveillance, establishes lifelong inflammation, predisposing to genomic instability and DNA damage, notably double strand breaks. The epithelial host cell responds by activation of DNA damage repair (DDR) machinery that seems to be compromised by the infection. It is therefore now accepted that genetic damage is a major mechanism operating in cases of H. pylori induced carcinogenesis. Here, we review the data on the molecular pathways involved in DNA damage and DDR activation during H. pylori infection. PMID:28289428

  16. Biological consequences of formation and repair of complex DNA damage.

    PubMed

    Magnander, Karin; Elmroth, Kecke

    2012-12-31

    Endogenous processes or genotoxic agents can induce many types of single DNA damage (single-strand breaks, oxidized bases and abasic sites). In addition, ionizing radiation induces complex lesions such as double-strand breaks and clustered damage. To preserve the genomic stability and prevent carcinogenesis, distinct repair pathways have evolved. Despite this, complex DNA damage can cause severe problems and is believed to contribute to the biological consequences observed in cells exposed to genotoxic stress. In this review, the current knowledge of formation and repair of complex DNA damage is summarized and the risks and biological consequences associated with their repair are discussed. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Pseudo-DNA damage response in senescent cells.

    PubMed

    Pospelova, Tatyana V; Demidenko, Zoya N; Bukreeva, Elena I; Pospelov, Valery A; Gudkov, Andrei V; Blagosklonny, Mikhail V

    2009-12-15

    Cellular senescence is currently viewed as a response to DNA damage. In this report, we showed that non-damaging agents such as sodium butyrate-induced p21 and ectopic expression of either p21 or p16 cause cellular senescence without detectable DNA breaks. Nevertheless, senescent cells displayed components of DNA damage response (DDR) such as gammaH2AX foci and uniform nuclear staining for p-ATM. Importantly, there was no accumulation of 53BP1 in gammaH2AX foci of senescent cells. Consistently, comet assay failed to detect DNA damage. Rapamycin, an inhibitor of mTO R, which was shown to suppress cellular senescence, decreased gammaH2AX foci formation. Thus, cellular senescence leads to activation of atypical DDR without detectable DNA damage. Pseudo-DDR may be a marker of general over-activation of senescent cells.

  18. 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

  19. An inducible long noncoding RNA amplifies DNA damage signaling.

    PubMed

    Schmitt, Adam M; Garcia, Julia T; Hung, Tiffany; Flynn, Ryan A; Shen, Ying; Qu, Kun; Payumo, Alexander Y; Peres-da-Silva, Ashwin; Broz, Daniela Kenzelmann; Baum, Rachel; Guo, Shuling; Chen, James K; Attardi, Laura D; Chang, Howard Y

    2016-11-01

    Long noncoding RNAs (lncRNAs) are prevalent genes with frequently precise regulation but mostly unknown functions. Here we demonstrate that lncRNAs guide the organismal DNA damage response. DNA damage activated transcription of the DINO (Damage Induced Noncoding) lncRNA via p53. DINO was required for p53-dependent gene expression, cell cycle arrest and apoptosis in response to DNA damage, and DINO expression was sufficient to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO bound to p53 protein and promoted its stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampened p53 signaling and ameliorated acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks.

  20. An inducible long noncoding RNA amplifies DNA damage signaling

    PubMed Central

    Schmitt, Adam M.; Garcia, Julia T.; Hung, Tiffany; Flynn, Ryan A.; Shen, Ying; Qu, Kun; Payumo, Alexander Y.; Peres-da-Silva, Ashwin; Broz, Daniela Kenzelmann; Baum, Rachel; Guo, Shuling; Chen, James K.; Attardi, Laura D.; Chang, Howard Y.

    2016-01-01

    Long noncoding RNAs (lncRNAs) are prevalent genes with frequently exquisite regulation but mostly unknown functions. Here we demonstrate a role of lncRNAs in guiding organismal DNA damage response. DNA damage activates transcription of DINO (Damage Induced NOncoding) via p53. DINO is required for p53-dependent gene expression, cell cycle arrest, and apoptosis in response to DNA damage, and DINO expression suffice to activate damage signaling and cell cycle arrest in the absence of DNA damage. DINO binds to and promotes p53 protein stabilization, mediating a p53 auto-amplification loop. Dino knockout or promoter inactivation in mice dampens p53 signaling and ameliorates acute radiation syndrome in vivo. Thus, inducible lncRNA can create a feedback loop with its cognate transcription factor to amplify cellular signaling networks. PMID:27668660

  1. Transcription and DNA Damage: Holding Hands or Crossing Swords?

    PubMed

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

    2016-11-05

    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. Published by Elsevier Ltd.

  2. Sodium tungstate modulates ATM function upon DNA damage.

    PubMed

    Rodriguez-Hernandez, C J; Llorens-Agost, M; Calbó, J; Murguia, J R; Guinovart, J J

    2013-05-21

    Both radiotherapy and most effective chemotherapeutic agents induce different types of DNA damage. Here we show that tungstate modulates cell response to DNA damaging agents. Cells treated with tungstate were more sensitive to etoposide, phleomycin and ionizing radiation (IR), all of which induce DNA double-strand breaks (DSBs). Tungstate also modulated the activation of the central DSB signalling kinase, ATM, in response to these agents. These effects required the functionality of the Mre11-Nbs1-Rad50 (MRN) complex and were mimicked by the inhibition of PP2A phosphatase. Therefore, tungstate may have adjuvant activity when combined with DNA-damaging agents in the treatment of several malignancies.

  3. DNA Damage Response and Autophagy: A Meaningful Partnership

    PubMed Central

    Eliopoulos, Aristides G.; Havaki, Sophia; Gorgoulis, Vassilis G.

    2016-01-01

    Autophagy and the DNA damage response (DDR) are biological processes essential for cellular and organismal homeostasis. Herein, we summarize and discuss emerging evidence linking DDR to autophagy. We highlight published data suggesting that autophagy is activated by DNA damage and is required for several functional outcomes of DDR signaling, including repair of DNA lesions, senescence, cell death, and cytokine secretion. Uncovering the mechanisms by which autophagy and DDR are intertwined provides novel insight into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and novel concepts for the development of improved therapeutic strategies against these pathologies. PMID:27917193

  4. Ultraviolet induced DNA damage and hereditary skin cancer

    SciTech Connect

    Regan, J.D.; Carrier, W.L.; Francis, A.A.

    1984-01-01

    Clearly, cells from normal individuals possess the ability to repair a variety of damage to DNA. Numerous studies indicate that defects in DNA repair may increase an individual's susceptibility to cancer. It is hoped that continued studies of the exact structural changes produced in the DNA by environmental insults, and the correlation of specific DNA changes with particulr cellular events, such as DNA repair, will lead to a better understanding of cell-killing, mutagenesis and carbinogenesis. 1 figure, 2 tables.

  5. Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair*

    PubMed Central

    Krasikova, Yuliya S.; Rechkunova, Nadejda I.; Maltseva, Ekaterina A.; Pestryakov, Pavel E.; Petruseva, Irina O.; Sugasawa, Kaoru; Chen, Xuejing; Min, Jung-Hyun; Lavrik, Olga I.

    2013-01-01

    The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (KD ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nm, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms. PMID:23443653

  6. Reshaping chromatin after DNA damage: the choreography of histone proteins.

    PubMed

    Polo, Sophie E

    2015-02-13

    DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information.

  7. Stress-induced DNA damage biomarkers: applications and limitations

    PubMed Central

    Nikitaki, Zacharenia; Hellweg, Christine E.; Georgakilas, Alexandros G.; Ravanat, Jean-Luc

    2015-01-01

    A variety of environmental stresses like chemicals, UV and ionizing radiation and organism's endogenous processes such as 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 damage plays 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 DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data. PMID:26082923

  8. Stress-induced DNA damage biomarkers: applications and limitations.

    PubMed

    Nikitaki, Zacharenia; Hellweg, Christine E; Georgakilas, Alexandros G; Ravanat, Jean-Luc

    2015-01-01

    A variety of environmental stresses like chemicals, UV and ionizing radiation and organism's endogenous processes such as 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 damage plays 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 DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data.

  9. DNA damage in kidney transplant patients. Role of organ origin.

    PubMed

    Corredor, Zuray; Rodríguez-Ribera, Lara; Coll, Elisabet; Silva, Irene; Díaz, Juan Manuel; Ballarín, José; Marcos, Ricard; Pastor, Susana

    2017-08-19

    Chronic kidney disease (CKD) patients are characterized by elevated levels of genomic damage. This damage increases when kidney function decreases being maximum in hemodialysis patients. As kidney transplantation improves renal function, and it is related with better survival, the aim of our study was to evaluate potential changes in DNA damage levels after kidney transplantation, and comparing living donor recipients with cadaveric donor recipients. The alkaline comet assay was used to determine DNA breaks and oxidative damaged DNA; and the micronucleus assay was used to determine chromosomal breakage and/or aneuploidy. Fifty CKD patients were followed up after 6 and 12 months of their kidney transplantation. All patients increased their genomic damage levels after 6 and 12 months of renal transplantation, compared with those observed before transplantation, despite of the improvement of their metabolic functions. Donor advanced age correlated positively with higher DNA damage. Genomic damage was lower in living donor transplants with respect to cadaveric donor transplants. Our conclusion is that DNA damage increased in kidney transplantation patients, whereas their renal function improved. Higher levels of DNA damage were found in cadaveric donor transplants when compared to living donor transplants. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. HDAC inhibitors: roles of DNA damage and repair.

    PubMed

    Robert, Carine; Rassool, Feyruz V

    2012-01-01

    Histone deacetylase inhibitors (HDACis) increase gene expression through induction of histone acetylation. However, it remains unclear whether specific gene expression changes determine the apoptotic response following HDACis administration. Herein, we discuss evidence that HDACis trigger in cancer and leukemia cells not only widespread histone acetylation but also actual increases in reactive oxygen species (ROS) and DNA damage that are further increased following treatment with DNA-damaging chemotherapies. While the origins of ROS production are not completely understood, mechanisms, including inflammation and altered antioxidant signaling, have been reported. While the generation of ROS is an explanation, at least in part, for the source of DNA damage observed with HDACi treatment, DNA damage can also be independently induced by changes in the DNA repair activity and chromatin remodeling factors. Recent development of sirtuin inhibitors (SIRTis) has shown that, similar to HDACis, these drugs induce increases in ROS and DNA damage used singly, or in combination with HDACis and other drugs. Thus, induction of apoptosis by HDACis/SIRTis may result through oxidative stress and DNA damage mechanisms in addition to direct activation of apoptosis-inducing genes. Nevertheless, while DNA damage and stress responses could be of interest as markers for clinical responses, they have yet to be validated as markers for responses to HDACi treatment in clinical trials, alone, and in combination.

  11. Chimeric Proteins to Detect DNA Damage and Mismatches

    SciTech Connect

    McCutchen-Maloney, S; Malfatti, M; Robbins, K M

    2002-01-14

    The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was demonstrated in

  12. Regulation of DNA Repair Mechanisms: How the Chromatin Environment Regulates the DNA Damage Response.

    PubMed

    Stadler, Jens; Richly, Holger

    2017-08-05

    Cellular DNA is constantly challenged by damage-inducing factors derived from exogenous or endogenous sources. In order to maintain genome stability and integrity, cells have evolved a wide variety of DNA repair pathways which counteract different types of DNA lesions, also referred to as the DNA damage response (DDR). However, DNA in eukaryotes is highly organized and compacted into chromatin representing major constraints for all cellular pathways, including DNA repair pathways, which require DNA as their substrate. Therefore, the chromatin configuration surrounding the lesion site undergoes dramatic remodeling to facilitate access of DNA repair factors and subsequent removal of the DNA lesion. In this review, we focus on the question of how the cellular DNA repair pathways overcome the chromatin barrier, how the chromatin environment is rearranged to facilitate efficient DNA repair, which proteins mediate this re-organization process and, consequently, how the altered chromatin landscape is involved in the regulation of DNA damage responses.

  13. Primary DNA Damage in Dry Cleaners with Perchlorethylene Exposure.

    PubMed

    Azimi, Mohammad; Bahrami, Mohammad Reza; Rezaei Hachesu, Vida; Zavar Reza, Javad; Mihanpour, Hamideh; Zare Sakhvidi, Mohammad Javad; Mostaghaci, Mehrdad

    2017-10-01

    Perchloroethylene is a halogenated solvent widely used in dry cleaning. International agency of research on cancer classified this chemical as a probable human carcinogen. To evaluate the extent of primary DNA damage in dry cleaner workers who were exposed to perchloroethylene as compared to non-exposed subjects. The effect of exposure modifying factors such as use of personal protective equipment, perceived risk, and reported safe behaviors on observed DNA damage were also studied. 59 exposed and non-exposed workers were selected from Yazd, Iran. All the 33 exposed workers had work history at least 3 months in the dry cleaning shops. Peripheral blood sampling was performed. Microscope examination was performed under fluorescent microscope (400×). Open comet software was used for image analysis. All biological analysis was performed in one laboratory. Primary DNA damage to leukocytes in dry cleaners was relatively high. The median tail length, %DNA in tail, and tail moment in exposed group were significantly higher than those in non-exposed group. There was no significant difference between smokers and nonsmokers in terms of tail length, tail moment, and %DNA in tail. There was no significant correlation between duration of employment in dry cleaning and observed DNA damage in terms of tail length, tail moment and %DNA in tail. Stratified analysis based on exposed and nonexposed category showed no significant relationship between age and observed DNA damage. Occupationally exposure to perchloroethylene can cause early DNA damage in dry cleaners.

  14. Single cell trapping and DNA damage analysis using microwell arrays

    PubMed Central

    Wood, David K.; Weingeist, David M.; Bhatia, Sangeeta N.; Engelward, Bevin P.

    2010-01-01

    With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health. PMID:20534572

  15. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  16. DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES

    EPA Science Inventory

    A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DN...

  17. DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES

    EPA Science Inventory

    A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DN...

  18. Chromatin perturbations during the DNA damage response in higher eukaryotes.

    PubMed

    Bakkenist, Christopher J; Kastan, Michael B

    2015-12-01

    The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response.

  19. DNA damage as a biological sensor for environmental sunlight.

    PubMed

    Schuch, André Passaglia; Garcia, Camila Carrião Machado; Makita, Kazuo; Menck, Carlos Frederico Martins

    2013-08-01

    Solar ultraviolet (UV) radiation is widely known as an environmental genotoxic agent that affects ecosystems and the human population, generating concerns and motivating worldwide scientific efforts to better understand the role of sunlight in the induction of DNA damage, cell death, mutagenesis, and ultimately, carcinogenesis. In this review, general aspects of UV radiation at the Earth's surface are reported, considering measurements by physical and biological sensors that monitor solar UV radiation under different environmental conditions. The formation of DNA photoproducts and other types of DNA damage by different UV wavelengths are compared with the present information on their roles in inducing biological effects. Moreover, the use of DNA-based biological dosimeters is presented as a feasible molecular and cellular tool that is focused on the evaluation of DNA lesions induced by natural sunlight. Clearly, direct environmental measurements demonstrate the biological impact of sunlight in different locations worldwide and reveal how this affects the DNA damage profile at different latitudes. These tools are also valuable for the quantification of photoprotection provided by commercial sunscreens against the induction of DNA damage and cell death, employing DNA repair-deficient cells that are hypersensitive to sunlight. Collectively, the data demonstrate the applicability of DNA-based biosensors as alternative, complementary, and reliable methods for registering variations in the genotoxic impact of solar UV radiation and for determining the level of photoprotection sunscreens provided at the level of DNA damage and cell death.

  20. 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.

  1. 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.

  2. Loss of Urokinase Receptor Sensitizes Cells to DNA Damage and Delays DNA Repair

    PubMed Central

    Narayanaswamy, Pavan B.; Hodjat, Mahshid; Haller, Hermann; Dumler, Inna; Kiyan, Yulia

    2014-01-01

    DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders. PMID:24987841

  3. Sperm DNA damage and its role in IVF and ICSI.

    PubMed

    Bach, Phil Vu; Schlegel, Peter N

    2016-01-01

    While the semen analysis has traditionally been relied upon to differentiate fertile and infertile men, its utility has been questioned in the current era of assisted reproductive technologies. The desire for more sophisticated diagnostic and predictive tools has led to increased use of sperm DNA damage in the management of male infertility. Despite the availability of numerous assays to measure sperm DNA damage, our understanding of the etiology, measurement, and clinical implications of sperm DNA damage remains incomplete. While the current evidence is fraught with heterogeneity that complicates attempts at comparison and meta-analysis, there does appear to be a role for sperm DNA damage in the development and maintenance of pregnancy in the era of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). However, as noted by the American Society for Reproductive Medicine, the routine and widespread use of sperm DNA damage testing is not yet supported. Further studies are needed to standardize the measurement of sperm DNA damage and to clarify the exact role of sperm DNA damage within the myriad of other male and female factors contributing to reproductive outcomes in IVF and ICSI.

  4. CHK2 kinase in the DNA damage response and beyond

    PubMed Central

    Zannini, Laura; Delia, Domenico; Buscemi, Giacomo

    2014-01-01

    The serine/threonine kinase CHK2 is a key component of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates >20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In particular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy. PMID:25404613

  5. Oxidative and non-oxidative DNA damage and cardiovascular disease.

    PubMed

    Malik, Qudsia; Herbert, Karl E

    2012-04-01

    Evidence for the association of DNA damage with cardiovascular disease has been obtained from in vitro cell culture models, experimental cardiovascular disease and analysis of samples obtained from humans with disease. There is general acceptance that several factors associated with the risk of developing cardiovascular disease cause oxidative damage to DNA in cell culture models with both nuclear and mitochondrial DNA as targets. Moreover, evidence obtained over the past 10 years points to a possible mechanistic role for DNA damage in experimental atherosclerosis culminating in recent studies challenging the assumption that DNA damage is merely a biomarker of the disease process. This kind of mechanistic insight provides a renewed impetus for further studies in this area.

  6. Imaging the DNA damage response with PET and SPECT.

    PubMed

    Knight, James C; Koustoulidou, Sofia; Cornelissen, Bart

    2017-06-01

    DNA integrity is constantly challenged by endogenous and exogenous factors that can alter the DNA sequence, leading to mutagenesis, aberrant transcriptional activity, and cytotoxicity. Left unrepaired, damaged DNA can ultimately lead to the development of cancer. To overcome this threat, a series of complex mechanisms collectively known as the DNA damage response (DDR) are able to detect the various types of DNA damage that can occur and stimulate the appropriate repair process. Each DNA damage repair pathway leads to the recruitment, upregulation, or activation of specific proteins within the nucleus, which, in some cases, can represent attractive targets for molecular imaging. Given the well-established involvement of DDR during tumorigenesis and cancer therapy, the ability to monitor these repair processes non-invasively using nuclear imaging techniques may facilitate the earlier detection of cancer and may also assist in monitoring response to DNA damaging treatment. This review article aims to provide an overview of recent efforts to develop PET and SPECT radiotracers for imaging of DNA damage repair proteins.

  7. 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.

  8. Single-step intercalating dye strategies for DNA damage studies.

    PubMed

    Paidipalli, Manasa; Pjescic, Ilija; Hindmarsh, Patrick L; Crews, Niel D

    2013-08-01

    Many analytical protocols exist for the quantification of varied types of DNA damage, which span a range of complexity and sensitivity. As an alternative or companion to existing procedures, this article demonstrates the application of quantitative PCR (qPCR) and high-resolution DNA melting analysis (HRMA) to the detection and quantification of intramolecular DNA damage and/or strand breaks. These proven molecular biology methods are essentially single-step processes. When implemented with a third-generation saturating DNA dye, high sensitivity can be obtained. The experiments presented here demonstrate how DNA damage can inhibit amplification of the affected molecules. This corresponding decrease in the initial concentration of amplifiable DNA can be measured with qPCR. In addition, damage in the form of intramolecular dimerization and strand breaks alters the stored energy in the hydrogen bonds between the two strands in the dsDNA molecule. This significantly affects the thermal stability, which can be measured with extreme precision using HRMA. Simplified damage models were used in these experiments: UV-C irradiation to produce photoproducts, and restriction enzyme digestion to simulate double-strand breaks. The findings of this work, however, can be intuitively applied to the broad scope of DNA damage mechanisms.

  9. 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.

  10. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    PubMed

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.

  11. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa

    PubMed Central

    Mahmoud, K. Gh. M.; El-Sokary, A. A. E.; Abdel-Ghaffar, A. E.; Abou El-Roos, M. E. A.; Ahmed, Y. F.

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  12. Reactive Scattering Damage to DNA Components by Hyperthermal Secondary Ions

    SciTech Connect

    Deng Zongwu; Bald, Ilko; Illenberger, Eugen; Huels, Michael A.

    2006-06-23

    We have observed reactive scattering damage to fundamental DNA building blocks by the type of hyperthermal secondary ions that are produced along heavy ion tracks in biological media. Reactions include carbon abstraction by N{sup +}, and hydrogen abstraction by O{sup -} and N{sup +}, at collision energies down to 1 eV. Our results show that localized reactive scattering by hyperthermal secondary fragments can lead to important physicochemical damage to DNA in cells irradiated by heavy ions. This suggests a fundamentally different picture of nascent DNA damage induced by heavy ion tracks, compared to conventional (x or {gamma}) radiation tracks.

  13. Reactive Scattering Damage to DNA Components by Hyperthermal Secondary Ions

    NASA Astrophysics Data System (ADS)

    Deng, Zongwu; Bald, Ilko; Illenberger, Eugen; Huels, Michael A.

    2006-06-01

    We have observed reactive scattering damage to fundamental DNA building blocks by the type of hyperthermal secondary ions that are produced along heavy ion tracks in biological media. Reactions include carbon abstraction by N+, and hydrogen abstraction by O- and N+, at collision energies down to 1 eV. Our results show that localized reactive scattering by hyperthermal secondary fragments can lead to important physicochemical damage to DNA in cells irradiated by heavy ions. This suggests a fundamentally different picture of nascent DNA damage induced by heavy ion tracks, compared to conventional (x or γ) radiation tracks.

  14. Inducible repair of oxidative DNA damage in Escherichia coli.

    PubMed

    Demple, B; Halbrook, J

    Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.

  15. Superoxide and the production of oxidative DNA damage.

    PubMed Central

    Keyer, K; Gort, A S; Imlay, J A

    1995-01-01

    The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron. PMID:7592468

  16. Radiation-induced DNA damage and chromatin structure

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

  17. DNA damage processing at telomeres: The ends justify the means.

    PubMed

    Fouquerel, Elise; Parikh, Dhvani; Opresko, Patricia

    2016-08-01

    Telomeres at chromosome ends are nucleoprotein structures consisting of tandem TTAGGG repeats and a complex of proteins termed shelterin. DNA damage and repair at telomeres is uniquely influenced by the ability of telomeric DNA to form alternate structures including loops and G-quadruplexes, coupled with the ability of shelterin proteins to interact with and regulate enzymes in every known DNA repair pathway. The role of shelterin proteins in preventing telomeric ends from being falsely recognized and processed as DNA double strand breaks is well established. Here we focus instead on recent developments in understanding the roles of shelterin proteins and telomeric DNA sequence and structure in processing genuine damage at telomeres induced by endogenous and exogenous DNA damage agents. We will highlight advances in double strand break repair, base excision repair and nucleotide excision repair at telomeres, and will discuss important questions remaining in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Radiation-induced DNA damage and chromatin structure

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

  19. A fluorescence enhancement assay for cellular DNA damage. [X Radiation

    SciTech Connect

    Kanter, P.M.; Schwartz, H.S.

    1982-07-01

    A fluorescence procedure is described for quantitative measurement of DNA damage in mammalian cells. The technique is based upon the time-dependent partial alkaline unwinding of cellular DNA followed by determination of duplex:total DNA ratios with bisbenzamide, which has a differential molar fluorescence with single-stranded and duplex DNA. The method is rapid, does not require radioactive labeling of DNA, and is sufficiently sensitive to detect damage induced with 100 rads of X-irradiation. This method is standardized with respect to the alkaline unwinding unit, Mn0, and the unwinding constant, beta. Results obtained with this new technique and with hydroxylapatite chromatography for physical separation of single- and double-stranded DNA were confirmatory. The utility of the technique was demonstrated by detection of dose-related damage with X-irradiation and a variety of antineoplastic agents in unlabeled murine leukemia cells.

  20. Oxidatively induced DNA damage and its repair in cancer.

    PubMed

    Dizdaroglu, Miral

    2015-01-01

    Oxidatively induced DNA damage is caused in living organisms by endogenous and exogenous reactive species. DNA lesions resulting from this type of damage are mutagenic and cytotoxic and, if not repaired, can cause genetic instability that may lead to disease processes including carcinogenesis. Living organisms possess DNA repair mechanisms that include a variety of pathways to repair multiple DNA lesions. Mutations and polymorphisms also occur in DNA repair genes adversely affecting DNA repair systems. Cancer tissues overexpress DNA repair proteins and thus develop greater DNA repair capacity than normal tissues. Increased DNA repair in tumors that removes DNA lesions before they become toxic is a major mechanism for development of resistance to therapy, affecting patient survival. Accumulated evidence suggests that DNA repair capacity may be a predictive biomarker for patient response to therapy. Thus, knowledge of DNA protein expressions in normal and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. DNA repair proteins constitute targets for inhibitors to overcome the resistance of tumors to therapy. Inhibitors of DNA repair for combination therapy or as single agents for monotherapy may help selectively kill tumors, potentially leading to personalized therapy. Numerous inhibitors have been developed and are being tested in clinical trials. The efficacy of some inhibitors in therapy has been demonstrated in patients. Further development of inhibitors of DNA repair proteins is globally underway to help eradicate cancer.

  1. DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response.

    PubMed

    Colis, Laureen; Peltonen, Karita; Sirajuddin, Paul; Liu, Hester; Sanders, Sara; Ernst, Glen; Barrow, James C; Laiho, Marikki

    2014-06-30

    DNA intercalation is a major therapeutic modality for cancer therapeutic drugs. The therapeutic activity comes at a cost of normal tissue toxicity and genotoxicity. We have recently described a planar heterocyclic small molecule DNA intercalator, BMH-21, that binds ribosomal DNA and inhibits RNA polymerase I (Pol I) transcription. Despite DNA intercalation, BMH-21 does not cause phosphorylation of H2AX, a key biomarker activated in DNA damage stress. Here we assessed whether BMH-21 activity towards expression and localization of Pol I marker proteins depends on DNA damage signaling and repair pathways. We show that BMH-21 effects on the nucleolar stress response were independent of major DNA damage associated PI3-kinase pathways, ATM, ATR and DNA-PKcs. However, testing a series of BMH-21 derivatives with alterations in its N,N-dimethylaminocarboxamide arm showed that several derivatives had acquired the property to activate ATM- and DNA-PKcs -dependent damage sensing and repair pathways while their ability to cause nucleolar stress and affect cell viability was greatly reduced. The data show that BMH-21 is a chemically unique DNA intercalator that has high bioactivity towards Pol I inhibition without activation or dependence of DNA damage stress. The findings also show that interference with DNA and DNA metabolic processes can be exploited therapeutically without causing DNA damage.

  2. Calculation of complex DNA damage induced by ions

    NASA Astrophysics Data System (ADS)

    Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

    2011-11-01

    This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

  3. Proteins in the Nutrient-Sensing and DNA Damage Checkpoint Pathways Cooperate to Restrain Mitotic Progression following DNA Damage

    PubMed Central

    Searle, Jennifer S.; Wood, Matthew D.; Kaur, Mandeep; Tobin, David V.; Sanchez, Yolanda

    2011-01-01

    Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA. PMID:21779180

  4. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  5. Bayesian estimation of sequence damage in ancient DNA.

    PubMed

    Ho, Simon Y W; Heupink, Tim H; Rambaut, Andrew; Shapiro, Beth

    2007-06-01

    DNA extracted from archaeological and paleontological remains is usually damaged by biochemical processes postmortem. Some of these processes lead to changes in the structure of the DNA molecule, which can result in the incorporation of incorrect nucleotides during polymerase chain reaction. These base misincorporations, or miscoding lesions, can lead to the inclusion of spurious additional mutations in ancient DNA (aDNA) data sets. This has the potential to affect the outcome of phylogenetic and population genetic analyses, including estimates of mutation rates and genetic diversity. We present a novel model, termed the delta model, which estimates the amount of damage in DNA data and accounts for its effects in a Bayesian phylogenetic framework. The ability of the delta model to estimate damage is first investigated using a simulation study. The model is then applied to 13 aDNA data sets. The amount of damage in these data sets is shown to be significant but low (about 1 damaged base per 750 nt), suggesting that precautions for limiting the influence of damaged sites, such as cloning and enzymatic treatment, are worthwhile. The results also suggest that relatively high rates of mutation previously estimated from aDNA data are not entirely an artifact of sequence damage and are likely to be due to other factors such as the persistence of transient polymorphisms. The delta model appears to be particularly useful for placing upper credibility limits on the amount of sequence damage in an alignment, and this capacity might be beneficial for future aDNA studies or for the estimation of sequencing errors in modern DNA.

  6. A model of the cell nucleus for DNA damage calculations.

    PubMed

    Nikjoo, Hooshang; Girard, Peter

    2012-01-01

    Development of a computer model of genomic deoxyribonucleic acid (DNA) in the human cell nucleus for DNA damage and repair calculations. The model comprises the human genomic DNA, chromosomal domains, and loops attached to factories. A model of canonical B-DNA was used to build the nucleosomes and the 30-nanometer solenoidal chromatin. In turn the chromatin was used to form the loops of factories in chromosome domains. The entire human genome was placed in a spherical nucleus of 10 micrometers diameter. To test the new target model, tracks of protons and alpha-particles were generated using Monte Carlo track structure codes PITS99 (Positive Ion Track Structure) and KURBUC. Damage sites induced in the genome were located and classified according to type and complexity. The three-dimensional structure of the genome starting with a canonical B-DNA model, nucleosomes, and chromatin loops in chromosomal domains are presented. The model was used to obtain frequencies of DNA damage induced by protons and alpha-particles by direct energy deposition, including single- and double-strand breaks, base damage, and clustered lesions. This three-dimensional model of the genome is the first such model using the full human genome for the next generation of more comprehensive modelling of DNA damage and repair. The model combines simple geometrical structures at the level of domains and factories with potentially full detail at the level of atoms in particular genes, allowing damage patterns in the latter to be simulated.

  7. DNA damage in cancer therapeutics: a boon or a curse?

    PubMed

    Khanna, Anchit

    2015-06-01

    Millions of DNA-damaging lesions occur every day in each cell of our bodies due to various stresses. The failure to detect and accurately repair these lesions can give rise to cells with high levels of endogenous DNA damage, deleterious mutations, or genomic aberrations. Such genomic instability can lead to the activation of specific signaling pathways, including the DNA damage response (DDR) pathway. Constitutive activation of DDR proteins has been observed in human tumor specimens from different cancer stages, including precancerous and metastatic cancers, although not in normal tissues. The tumor-suppressive role of DDR activity during the premalignant stage has been studied, and strong evidence is emerging for an oncogenic role for DDR proteins such as DNA-PK and CHK1 during the later stages of tumor development. However, the majority of current cancer therapies induce DNA damage, potentially exacerbating protumorigenic genomic instability and enabling the development of resistance. Therefore, elucidating the molecular basis of DNA damage-mediated genomic instability and its role in tumorigenesis is critical. Finally, I discuss the potential existence of distinct DNA damage thresholds at various stages of tumorigenesis and what the ramifications of such thresholds would be, including the ambiguous role of the DDR pathway in human cancers, therapy-induced malignancies, and enhanced therapies. ©2015 American Association for Cancer Research.

  8. Commentary: Mitochondrial DNA damage and loss in diabetes.

    PubMed

    Gilkerson, Robert

    2016-10-01

    This commentary discusses damage and loss of mitochondrial DNA (mtDNA) in type 2 diabetes mellitus from both the clinical and experimental perspectives. Increasingly, an array of studies in experimental models and patients suggests that the cellular stresses of insulin resistance in type 2 diabetes damage mtDNA, leading to loss of mitochondrial genetic content. As such, mtDNA is emerging as both a valuable monitoring tool and translational preventive target for metabolic disease. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Damage-specific DNA-binding proteins from human cells

    SciTech Connect

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1% NP-40.

  10. Electrochemical study of DNA damaged by oxidation stress.

    PubMed

    Zitka, Ondrej; Krizkova, Sona; Skalickova, Sylvie; Kopel, Pavel; Babula, Petr; Adam, Vojtech; Kizek, Rene

    2013-02-01

    Many compounds can interact with DNA leading to changes of DNA structure as point mutation and bases excision, which could trigger some metabolic failures, which leads to the changes in DNA structure resulting in cancer. Oxidation of nucleic acid bases belongs to the one of the mostly occurred type of DNA damaging leading to the above mentioned phenomena. The investigation of processes of DNA oxidation damage is topical and electrochemical methods include a versatile and sensitive tool for these purposes. 8-hydroxydeoxyguanosine (8-OHdG) is the most widely accepted marker of DNA damage. Oxidative damage to DNA by free radicals and exposure to ionizing radiation generate several other products within the double helix besides mentioned oxidation products of nucleic acid bases. The basic electrochemical behaviour of nucleic acids bases on various types of carbon electrodes is reviewed. Further, we address our attention on description of oxidation mechanisms and on detection of the most important products of nucleic bases oxidation. The miniaturization of detector coupled with some microfluidic devices is suggested and discussed. The main aim of this review is to report the advantages and features of the electrochemical detection of guanine oxidation product as 8-OHdG and other similarly produced molecules as markers for DNA damage.

  11. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  12. ISWI chromatin remodeling complexes in the DNA damage response.

    PubMed

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA.

  13. DNA damage in Wistar Kyoto rats exercised during pregnancy.

    PubMed

    Corrêa, Mikaela da Silva; Gelaleti, Rafael Bottaro; Bento, Giovana Fernanda; Damasceno, Débora Cristina; Peraçoli, José Carlos

    2017-05-01

    To evaluate DNA damage levels in pregnant rats undergoing a treadmill exercise program. Wistar Kyoto rats were allocated into two groups (n= 5 animals/group): non-exercise and exercise. The pregnant rats were underwent an exercise protocol on a treadmill throughout pregnancy. Exercise intensity was set at 50% of maximal capacity during maximal exercise testing performed before mating. Body weight, blood pressure and glucose levels, and triglyceride concentration were measured during pregnancy. At day 10 post-natal, the animals were euthanized and maternal blood samples were collected for DNA damage. Blood pressure and glucose levels and biochemical measurements showed no significant differences. Increased DNA damage levels were found in exercise group compared to those of non-exercise group (p<0.05). The exercise intensity protocol used in the study might have been exhaustive leading to maternal increased DNA damage levels, demonstrating the relevance of an adequate protocol of physical exercise.

  14. Carbamate insecticide methomyl confers cytotoxicity through DNA damage induction.

    PubMed

    Guanggang, Xiang; Diqiu, Li; Jianzhong, Yuan; Jingmin, Guan; Huifeng, Zhai; Mingan, Shi; Liming, Tao

    2013-03-01

    Carbamate insecticide methomyl could induce genotoxic effects, including micronuclei, chromosome aberrations and sister-chromatid exchanges. However, methomyl induction of cytotoxicity through DNA damage is largely unknown. Here we identify cytotoxicity and potential genotoxicity of methomyl in vitro. We have employed alkaline comet assay, γH2AX foci formation and DNA ladder assay to detected DNA damage and apoptosis of Drosophila S2, HeLa and HEK293 cells. The alkaline comet assay was used to evaluate total DNA single strand breaks (SSBs) in the target cells exposed in vitro to sublethal concentrations of methomyl. As expected, methomyl induced significant concentration-dependent increases in DNA damage of target cells compared with the negative control, as measured by increases in tail length (μm), tail DNA (percentage of the comet tail) and tail moment (arbitrary units). In agreement with the comet assay data, the percentage of γH2AX positive reaction in HeLa cells also revealed methomyl caused DNA double strand breaks (DSBs) in a time-dependent manner. Moreover, methomyl induced a significant increase of apoptosis in Drosophila S2, HeLa and HEK293 cells in a concentration- and time-dependent manner, as determined by Urea PAGE DNA fragmentation analysis. In conclusion, methomyl is a strongly genotoxic agent that induces cell DNA damage and apoptosis in vitro at these sublethal concentrations.

  15. 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

  16. 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.

  17. Roles of RNA-Binding Proteins in DNA Damage Response.

    PubMed

    Kai, Mihoko

    2016-02-27

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  18. Chemical studies on DNA damage by radiation

    NASA Astrophysics Data System (ADS)

    Kagiya, V. Tsutomu; Sakano, Koichi; Nishimoto, Sei-Ici

    The radiation chemical characteristics of DNA-related compounds, especially thymine, as the most radiosensitivity DNA base in aqueous solution, are shown to obtain correlations with the radiation inactivations of bacterial and mammalian cultured cells. Recent development of chemical sensitizers for modification of the radiosensitivity of hypoxic cells, which is closely associated with the radiation therapy of cancer, is also reviewed.

  19. Oxidative DNA damage and repair in teratogenesis and neurodevelopmental deficits.

    PubMed

    Wells, Peter G; McCallum, Gordon P; Lam, Kyla C H; Henderson, Jeffrey T; Ondovcik, Stephanie L

    2010-06-01

    Several teratogenic agents, including ionizing radiation and xenobiotics such as phenytoin, benzo[a]pyrene, thalidomide, and methamphetamine, can initiate the formation of reactive oxygen species (ROS) that oxidatively damage cellular macromolecules including DNA. Oxidative DNA damage, and particularly the most prevalent 8-oxoguanine lesion, may adversely affect development, likely via alterations in gene transcription rather than via a mutational mechanism. Contributions from oxidative DNA damage do not exclude roles for alternative mechanisms of initiation like receptor-mediated processes or the formation of covalent xenobiotic-macromolecular adducts, damage to other macromolecular targets like proteins and lipids, and other effects of ROS like altered signal transduction. Even in the absence of teratogen exposure, endogenous developmental oxidative stress can have embryopathic consequences in the absence of key pathways for detoxifying ROS or repairing DNA damage. Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not always, protect the embryo from ROS-initiating teratogens. Protection may be variably dependent upon such factors as the nature of the teratogen and its concentration within the embryo, the stage of development, the species, strain, gender, target tissue and cell type, among other factors.

  20. Prevention of DNA damage in human skin by topical sunscreens.

    PubMed

    Olsen, Catherine M; Wilson, Louise F; Green, Adèle C; Biswas, Neela; Loyalka, Juhi; Whiteman, David C

    2017-05-01

    There is strong evidence that topical sunscreens, designed to protect against ultraviolet radiation (UVR)-induced erythema, decrease the amount of UVR to which the skin is exposed, but their effectiveness in reducing UVR-induced DNA damage in vivo has not been well quantified. We systematically reviewed the published literature (1990-2015) to determine whether sunscreens prevent DNA damage in human skin when applied prior to UVR exposure. We included experimental studies measuring UVR-induced DNA damage in human skin in vivo with and without sunscreens and excluded studies conducted in animal models and cell lines. Eligible studies were identified by computerized search of bibliographic databases, supplemented by hand-searching the reference lists of retrieved articles. We identified ten eligible studies. Despite heterogeneity in methodological approaches, including the sun protection factors of the sunscreens assessed, range of skin types examined, the UVR exposure time and dose, the timing of post-irradiation biopsies and in the markers of DNA damage examined, all studies reported markedly reduced (or nil) UVR-induced DNA damage on sunscreen-protected skin. Our review of the experimental evidence supports a protective effect of topical sunscreens in preventing UVR-induced DNA damage in human skin cells in vivo. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. 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.

  2. Spatially localized generation of nucleotide sequence-specific DNA damage

    PubMed Central

    Oh, Dennis H.; King, Brett A.; Boxer, Steven G.; Hanawalt, Philip C.

    2001-01-01

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen–DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320–400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA–psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen–TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues. PMID:11572980

  3. Aberrant BLM cytoplasmic expression associates with DNA damage stress and hypersensitivity to DNA-damaging agents in colorectal cancer.

    PubMed

    Votino, Carolina; Laudanna, Carmelo; Parcesepe, Pietro; Giordano, Guido; Remo, Andrea; Manfrin, Erminia; Pancione, Massimo

    2017-03-01

    Bloom syndrome is a rare and recessive disorder characterized by loss-of-function mutations of the BLM gene, which encodes a RecQ 3'-5' DNA helicase. Despite its putative tumor suppressor function, the contribution of BLM to human sporadic colorectal cancer (CRC) remains poorly understood. The transcriptional regulation mechanism underlying BLM and related DNA damage response regulation in independent CRC subsets and a panel of derived cell lines was investigated by bioinformatics analysis, the transcriptomic profile, a CpG island promoter methylation assay, Western blot, and an immunolocalization assay. In silico analysis of gene expression data sets revealed that BLM is overexpressed in poorly differentiated CRC and exhibits a close connection with shorter relapse-free survival even after adjustment for prognostic factors and pathways that respond to DNA damage response through ataxia telangiectasia mutated (ATM) signaling. Functional characterization demonstrated that CpG island promoter hypomethylation increases BLM expression and associates with cytoplasmic BLM mislocalization and increased DNA damage response both in clinical CRC samples and in derived cancer cell lines. The DNA-damaging agent S-adenosylmethionine suppresses BLM expression, leading to the inhibition of cell growth following accumulation of DNA damage. In tumor specimens, cytoplasmic accumulation of BLM correlates with DNA damage and γH2AX and phosphorylated ATM foci and predicts long-term progression-free survival in metastatic patients treated with irinotecan. Taken together, the findings of this study provide the first evidence that cancer-linked DNA hypomethylation and cytosolic BLM mislocalization might reflect compromised levels of DNA-repair activity and enhanced hypersensitivity to DNA-damaging agents in CRC patients.

  4. The Cartography of UV-induced DNA Damage Formation and DNA Repair.

    PubMed

    Hu, Jinchuan; Adar, Sheera

    2017-01-01

    DNA damage presents a barrier to DNA-templated biochemical processes, including gene expression and faithful DNA replication. Compromised DNA repair leads to mutations, enhancing the risk for genetic diseases and cancer development. Conventional experimental approaches to study DNA damage required a researcher to choose between measuring bulk damage over the entire genome, with little or no resolution regarding a specific location, and obtaining data specific to a locus of interest, without a global perspective. Recent advances in high-throughput genomic tools overcame these limitations and provide high-resolution measurements simultaneously across the genome. In this review, we discuss the available methods for measuring DNA damage and their repair, focusing on genomewide assays for pyrimidine photodimers, the major types of damage induced by ultraviolet irradiation. These new genomic assays will be a powerful tool in identifying key components of genome stability and carcinogenesis. © 2016 The American Society of Photobiology.

  5. DNA damage in mouse lymphocytes exposed to curcumin and copper.

    PubMed

    Urbina-Cano, Patricia; Bobadilla-Morales, Lucina; Ramírez-Herrera, Mario A; Corona-Rivera, Jorge R; Mendoza-Magaña, Maria L; Troyo-Sanromán, Rogelio; Corona-Rivera, Alfredo

    2006-01-01

    Dietary polyphenolics, such as curcumin, have shown antioxidant and anti-inflammatory effects. Some antioxidants cause DNA strand breaks in excess of transition metal ions, such as copper. The aim of this study was to evaluate the in vitro effect of curcumin in the presence of increasing concentrations of copper to induce DNA damage in murine leukocytes by the comet assay. Balb-C mouse lymphocytes were exposed to 50 microM curcumin and various concentrations of copper (10 microM, 100 microM and 200 microM). Cellular DNA damage was detected by means of the alkaline comet assay. Our results show that 50 microM curcumin in the presence of 100-200 microM copper induced DNA damage in murine lymphocytes. Curcumin did not inhibit the oxidative DNA damage caused by 50 microM H2O2 in mouse lymphocytes. Moreover, 50 microM curcumin alone was capable of inducing DNA strand breaks under the tested conditions. The increased DNA damage by 50 mM curcumin was observed in the presence of various concentrations of copper, as detected by the alkaline comet assay.

  6. Single-molecule visualization of ROS-induced DNA damage in large DNA molecules.

    PubMed

    Lee, Jinyong; Kim, Yongkyun; Lim, Sangyong; Jo, Kyubong

    2016-02-07

    We present a single molecule visualization approach for the quantitative analysis of reactive oxygen species (ROS) induced DNA damage, such as base oxidation and single stranded breaks in large DNA molecules. We utilized the Fenton reaction to generate DNA damage with subsequent enzymatic treatment using a mixture of three types of glycosylases to remove oxidized bases, and then fluorescent labeling on damaged lesions via nick translation. This single molecule analytical platform provided the capability to count one or two damaged sites per λ DNA molecule (48.5 kb), which were reliably dependent on the concentrations of hydrogen peroxide and ferrous ion at the micromolar level. More importantly, the labeled damaged sites that were visualized under a microscope provided positional information, which offered the capability of comparing DNA damaged sites with the in silico genomic map to reveal sequence specificity that GTGR is more sensitive to oxidative damage. Consequently, single DNA molecule analysis provides a sensitive analytical platform for ROS-induced DNA damage and suggests an interesting biochemical insight that the genome primarily active during the lysogenic cycle may have less probability for oxidative DNA damage.

  7. MECHANISTIC AND BIOLOGICAL ASPECTS OF HELICASE ACTION ON DAMAGED DNA

    PubMed Central

    Suhasini, Avvaru N.; Brosh, Robert M.

    2010-01-01

    Helicases catalytically unwind structured nucleic acids in a nucleoside-triphosphate-dependent and directionally specific manner, and are essential for virtually all aspects of nucleic acid metabolism. ATPase-driven helicases which translocate along nucleic acids play a role in damage recognition or unwinding of a DNA tract containing the lesion. Although classical biochemical experiments provided evidence that bulky covalent adducts inhibit DNA unwinding catalyzed by certain DNA helicases in a strand-specific manner (i.e. , block to DNA unwinding restricted to adduct residence in the strand the helicase translocates), recent studies suggest more complex arrangements that may depend on the helicase under study, its assembly in a protein complex, and the type of structural DNA perturbation. Moreover, base and sugar phosphate backbone modifications exert effects on DNA helicases that suggest specialized tracking mechanisms. As a component of the replication stress response, the single-stranded DNA binding protein Replication Protein A (RPA) may serve to enable eukaryotic DNA helicases to overcome certain base lesions. Helicases play important roles in DNA damage signaling which also involve their partnership with RPA. In this review, we will discuss our current understanding of mechanistic and biological aspects of helicase action on damaged DNA. PMID:20574162

  8. Insight into the ERVK Integrase – Propensity for DNA Damage

    PubMed Central

    Bray, Samantha; Turnbull, Matthew; Hebert, Sherry; Douville, Renée N.

    2016-01-01

    Retroviruses create permanently integrated proviruses that exist in the host genome. Retroviral genomes encode for functionally conserved gag, pro, pol, and env regions, as well as integrase (IN), which is required for retroviral integration. IN mediates viral genome insertion through 3′ end processing of the viral DNA and the strand transfer reaction. This process requires the formation of a pre-integration complex, comprised of IN, viral DNA, and cellular proteins. Viral insertion causes DNA damage, leading to the requirement of host DNA repair mechanisms. Therefore, a failure of DNA repair pathways may result in genomic instability and potentially cause host cell death. Considering the numerous human diseases associated with genomic instability, the endogenous retrovirus-K (ERVK) IN should be considered as a putative contributor to DNA damage in human cells. Future research and drug discovery should focus on ERVK IN activity and its role in human conditions, such as neurological disease and cancers. PMID:27990140

  9. 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.

  10. Curcumin Triggers DNA Damage and Inhibits Expression of DNA Repair Proteins in Human Lung Cancer Cells.

    PubMed

    Ting, Chien-Yi; Wang, Hsin-Ell; Yu, Chien-Chih; Liu, Hsin-Chung; Liu, Yu-Chang; Chiang, I-Tsang

    2015-07-01

    The study goal was to evaluate the effects of curcumin on DNA damage and expression of DNA-repair proteins in human lung cancer. Thus, NCI-H460 cells were used to study the effects of curcumin on DNA damage and repair in vitro. We investigated curcumin induces DNA damage by comet the assay and 4',6-diamidino-2-phenylindole (DAPI) staining. The DNA damage/repair-related protein levels were examined and monitored by western blotting and confocal microscopy. Curcumin significantly increased the length of comet tails and DNA condensation in NCI-H460 cells. Curcumin reduced expression of DNA-repair proteins such as 14-3-3 protein sigma (14-3-3σ), O6-methylguanine-DNA methyltransferase (MGMT), breast cancer susceptibility gene 1 (BRCA1), and mediator of DNA damage checkpoint 1 (MDC1). Curcumin also increased phosphorylation of p53 and Histone H2A.X (S140) in the nuclei of NCI-H460 cells. Taken together, our findings indicated that curcumin triggered DNA damage and inhibited expression of DNA-repair-associated proteins in NCI-H460 cells. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  11. DNA damage response to the Mdm2 inhibitor Nutlin-3

    PubMed Central

    Verma, Rajeev; Rigatti, Marc J.; Belinsky, Glenn S.; Godman, Cassandra A.; Giardina, Charles

    2009-01-01

    Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM0 colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin). Quantification of γH2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a ~50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events. PMID:19788889

  12. The intersection between DNA damage response and cell death pathways.

    PubMed

    Nowsheen, S; Yang, E S

    2012-10-01

    Apoptosis is a finely regulated process that serves to determine the fate of cells in response to various stresses. One such stress is DNA damage, which not only can signal repair processes but is also intimately involved in regulating cell fate. In this review we examine the relationship between the DNA damage/repair response in cell survival and apoptosis following insults to the DNA. Elucidating these pathways and the crosstalk between them is of great importance, as they eventually contribute to the etiology of human disease such as cancer and may play key roles in determining therapeutic response. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

  13. 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

  14. DNA damage in dihydroartemisinin-resistant Molt-4 cells.

    PubMed

    Park, Jungsoo; Lai, Henry C; Sasaki, Tomikazu; Singh, Narendra P

    2015-03-01

    Artemisinin generates carbon-based free radicals when it reacts with iron, and induces molecular damage and apoptosis. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular free iron. Dihydroartemisinin (DHA), an analog of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A major concern is whether cancer cells could develop resistance to DHA, thus limiting its therapeutic efficacy. We have developed a DHA-resistant Molt-4 cell line (RTN) and found out that these cells exhibited resistance to DHA but no significant cross- resistance to artemisinin-tagged holotransferrin (ART-TF), a synthetic artemisinin compound. In the present study, we investigated DNA damage induced by DHA and ART-TF in both Molt-4 and RTN cells using the comet assay. RTN cells exhibited a significantly lower level of basal and X-ray-induced DNA damage compared to Molt-4 cells. Both DHA and ART-TF induced DNA damage in Molt-4 cells, whereas DNA damage was induced in RTN cells by ART-TF, and not DHA. The result of this study shows that by the cell selection method, it is possible to generate a Molt-4 cell line which is not sensitive to DHA, but sensitive to ART-TF, as measured by DNA damage.

  15. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors.

    PubMed

    Francia, Sofia; Cabrini, Matteo; Matti, Valentina; Oldani, Amanda; d'Adda di Fagagna, Fabrizio

    2016-04-01

    The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling.

  16. Synthesis of damaged DNA containing the oxidative lesion 3'-oxothymidine.

    PubMed

    Bedi, Mel F; Li, Weiye; Gutwald, Taylor; Bryant-Friedrich, Amanda C

    2017-09-01

    Oxidative events that take place during regular oxygen metabolism can lead to the formation of organic or inorganic radicals. The interaction of these radicals with macromolecules in the organism and with DNA in particular is suspected to lead to apoptosis, DNA lesions and cell damage. Independent generation of DNA lesions resulting from oxidative damage is used to promote the study of their effects on biological systems. An efficient synthesis of oligodeoxyribonucleotides (ODNs) containing the oxidative damage lesion 3'-oxothymidine has been accomplished via incorporation of C3'-hydroxymethyl thymidine as its corresponding 5'-phosphoramidite. Through oxidative cleavage using sodium periodate in aqueous solution, the lesion of interest is easily generated. Due to its inherent instability it cannot be directly isolated, but must be generated in situ. 3'-Oxothymidine is a demonstrated damage product formed upon generation of the C3'-thymidinyl radical in ODN. Copyright © 2017. Published by Elsevier Ltd.

  17. DNA photo-oxidative damage hazard in transfection complexes.

    PubMed

    Rudiuk, Sergii; Franceschi-Messant, Sophie; Chouini-Lalanne, Nadia; Perez, Emile; Rico-Lattes, Isabelle

    2011-01-01

    Complexes of DNA with various cationic vectors have been largely used for nonviral transfection, and yet the photochemical stability of DNA in such complexes has never been considered. We studied, for the first time, the influence of DNA complexation by a cationic lipid and polymers on the amount of damage induced by benzophenone photosensitization. The localization of benzophenone inside the hydrophobic domains formed by a cationic lipid, DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and close to DNA, locally increases the photoinduced cleavage by the reactive oxygen species generated. The same effect was found in the case of DNA complexation with an amphiphilic polymer (polynorbornenemethyleneammonium chloride). However, a decrease in DNA damage was observed in the case of complexation with a hydrophilic polymer (polyethylenimine). The DNA protection in this case was because of the absence of benzophenone hydrophobic incorporation into the complex, and to DNA compaction which decreased the probability of radical attack. These results underline the importance of the chemical structure of the nonviral transfection vector in limiting the risks of photo-oxidative damage of the complexed DNA. © 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.

  18. Systemic DNA damage responses in aging and diseases.

    PubMed

    Ribezzo, Flavia; Shiloh, Yosef; Schumacher, Björn

    2016-06-01

    The genome is constantly attacked by a variety of genotoxic insults. The causal role for DNA damage in aging and cancer is exemplified by genetic defects in DNA repair that underlie a broad spectrum of acute and chronic human disorders that are characterized by developmental abnormalities, premature aging, and cancer predisposition. The disease symptoms are typically tissue-specific with uncertain genotype-phenotype correlation. The cellular DNA damage response (DDR) has been extensively investigated ever since yeast geneticists discovered DNA damage checkpoint mechanisms, several decades ago. In recent years, it has become apparent that not only cell-autonomous but also systemic DNA damage responses determine the outcome of genome instability in organisms. Understanding the mechanisms of non-cell-autonomous DNA damage responses will provide important new insights into the role of genome instability in human aging and a host of diseases including cancer and might better explain the complex phenotypes caused by genome instability. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. DNA damage caused by pesticide-contaminated soil.

    PubMed

    Krishnamurthi, K; Saravana Devi, S; Chakrabarti, T

    2006-12-01

    To determine the DNA damaging potential and the genotoxicity of individual compounds in pesticide contaminated soil. In the present study, DNA damaging potential of pesticide-contaminated soil and the genotoxicity of individual compounds present in the soil were assessed using fluorimetric analysis of DNA unwinding assay. The contaminated soil sample showed 79% (P < 0.001) of DNA strand break, whereas technical grade of major carbaryl and alpha-naphthol constituents of the contaminated soil showed 64% (P < 0.01) and 60% (P < 0.02) damage respectively. Our results indicate that the toxicity caused by contaminated soil is mainly due to carbaryl and alpha-napthol, which are the major constituents of the soil sample analyzed by GC-MS.

  20. Investigating DNA Radiation Damage Using X-Ray Absorption Spectroscopy

    PubMed Central

    Czapla-Masztafiak, Joanna; Szlachetko, Jakub; Milne, Christopher J.; Lipiec, Ewelina; Sá, Jacinto; Penfold, Thomas J.; Huthwelker, Thomas; Borca, Camelia; Abela, Rafael; Kwiatek, Wojciech M.

    2016-01-01

    The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA. PMID:27028640

  1. Bacterial natural transformation by highly fragmented and damaged DNA.

    PubMed

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A A; Mayar, J Victor Moreno; Rasmussen, Simon; Dahl, Tais W; Rosing, Minik T; Poole, Anthony M; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-12-03

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥ 20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.

  2. Oxidative DNA Damage in Blood of CVD Patients Taking Detralex

    PubMed Central

    Krzyściak, Wirginia; Cierniak, Agnieszka; Kózka, Mariusz; Kozieł, Joanna

    2011-01-01

    The main goal of the work reported here was to determine the degree of oxidative/alkali-labile DNA damages in peripheral blood as well as in the blood stasis from varicose vein of (chronic venous disorder) CVD patients. Moreover, determination of the impact of Detralex usage on the level of (oxidative) DNA damages in CVD patients was evaluated as well. The degree of oxidative DNA damages was studied in a group consisted of thirty patients with diagnosed chronic venous insufficiency (CVI) in the 2nd and 3rd degree, according to clinical state, etiology, anatomy and pathophysiology (CEAP), and qualified to surgical procedure. The control group consisted of normal volunteers (blood donors) qualified during standard examinations at Regional Centers of Blood Donation and Blood Therapy. The comet assay was used for determination of DNA damages. Analyses of the obtained results showed increase in the level of oxidative/alkali-labile DNA damages in lymphocytes originating from antebrachial blood of CVD patients as compared to the control group (Control) (p < 0.002; ANOVA). In addition, it was demonstrated that the usage of Detralex® resulted in decrease of the level of oxidative/alkali-labile DNA damages in CVD patients as compared to patients without Detralex® treatment (p < 0.001; ANOVA). Based on findings from the study, it may be hypothesized about occurrence of significant oxidative DNA damages as the consequence of strong oxidative stress in CVD. In addition, antioxidative effectiveness of Detralexu® was observed at the recommended dose, one tablet twice daily. PMID:21912579

  3. Tyrosine-dependent oxidative DNA damage induced by carcinogenic tetranitromethane.

    PubMed

    Murata, Mariko; Kurimoto, Saori; Kawanishi, Shosuke

    2006-10-01

    Tetranitromethane (TNM) is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel. TNM was reported to induce pulmonary adenocarcinomas and squamous cell carcinomas in mice and rats. However, the mechanisms underlying carcinogenesis induced by TNM has not yet been clarified. We previously revealed that nitroTyr and nitroTyr-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Since TNM is a reagent for nitration of Tyr in proteins and peptides, we have hypothesized that TNM-treated Tyr and Tyr-containing peptides induce DNA damage by the modification of Tyr. We examined DNA damage induced by TNM-treated amino acids or peptides using (32)P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. TNM-treated Tyr and Lys-Tyr-Lys induced DNA damage including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of Cu(II) and NADH. DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). The cytosine residue of the ACG sequence complementary to codon 273, well-known hotspots of the p53 gene, was cleaved with piperidine and Fpg treatments. On the other hand, nitroTyr and Lys-nitroTyr-Lys did not induce DNA damage in the presence of Cu(II) and NADH. Time-of-flight mass spectrometry confirmed that reactions between Lys-Tyr-Lys and TNM yielded not only Lys-nitroTyr-Lys but also Lys-nitrosoTyr-Lys. Therefore, it is speculated that the nitrosotyrosine residue can induce oxidative DNA damage in the presence of Cu(II) and NADH. It is concluded that Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM. TNM is a new type of carcinogen that induces DNA damage not by itself but via Tyr modification.

  4. Oxidative DNA damage during sleep periods among nightshift workers.

    PubMed

    Bhatti, Parveen; Mirick, Dana K; Randolph, Timothy W; Gong, Jicheng; Buchanan, Diana Taibi; Zhang, Junfeng Jim; Davis, Scott

    2016-08-01

    Oxidative DNA damage may be increased among nightshift workers because of suppression of melatonin, a cellular antioxidant, and/or inflammation related to sleep disruption. However, oxidative DNA damage has received limited attention in previous studies of nightshift work. From two previous cross-sectional studies, urine samples collected during a night sleep period for 217 dayshift workers and during day and night sleep (on their first day off) periods for 223 nightshift workers were assayed for 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, using high-performance liquid chromatography with electrochemical detection. Urinary measures of 6-sulfatoxymelatonin (aMT6s), a marker of circulating melatonin levels, and actigraphy-based sleep quality data were also available. Nightshift workers during their day sleep periods excreted 83% (p=0.2) and 77% (p=0.03) of the 8-OH-dG that dayshift workers and they themselves, respectively, excreted during their night sleep periods. Among nightshift workers, higher aMT6s levels were associated with higher urinary 8-OH-dG levels, and an inverse U-shaped trend was observed between 8-OH-dG levels and sleep efficiency and sleep duration. Reduced excretion of 8-OH-dG among nightshift workers during day sleep may reflect reduced functioning of DNA repair machinery, which could potentially lead to increased cellular levels of oxidative DNA damage. Melatonin disruption among nightshift workers may be responsible for the observed effect, as melatonin is known to enhance repair of oxidative DNA damage. Quality of sleep may similarly impact DNA repair. Cellular levels of DNA damage will need to be evaluated in future studies to help interpret these findings. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  5. Aven-dependent activation of ATM following DNA damage

    PubMed Central

    Guo, Jessie Yanxiang; Yamada, Ayumi; Kajino, Taisuke; Wu, Judy Qiju; Tang, Wanli; Freel, Christopher D.; Feng, Junjie; Chau, B. Nelson; Wang, Michael Zhuo; Margolis, Seth; Yoo, Hae Yong; Wang, Xiao-Fan; Dunphy, William G.; Irusta, Pablo M.; Hardwick, J. Marie; Kornbluth, Sally

    2009-01-01

    Summary Background In response to DNA damage, cells either undergo cell cycle arrest or apoptosis, depending on the extent of damage and the cell’s capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors such as Chk2 and p53 to inhibit cell cycle progression. ATM is recruited to double stranded DNA breaks by a complex of sensor proteins including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. Results In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpression in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knock-down of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM following DNA damage is enhanced by ATM-mediated Aven phosphorylation. Conclusions These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA damage signal. PMID:18571408

  6. Fluorescent reports for detection and measurement of DNA damage

    SciTech Connect

    Uziel, M.; Houck, K. )

    1993-01-01

    Epidemiological studies of real populations are complicated by the inevitable coexistence of exposure to multiple agents within the target population. An alternative method for characterizing these types of exposures is to use the reactive chemical functional group as the toxic agent identify the corresponding classes (families) of damage as markers of effects. We have begun studies to develop spectrometric reporters of DNA damage that can be measured on intact DNA. The direct measurement of adducts on microgram levels of DNA from tissue biopsy may succeed because of the high sensitivity and selectivity of different reporter compounds. While one cannot readily distinguish between recent or persistent exposures, baseline values for individuals may be constructed. For example, normal oxidative metabolism and environmental radiation create oxidation processes that continually damage DNA. These reactions create lesions that can be measured with the reporter compound FABA [N- (5- fluoresceinyl), N[prime]-(3-boronatophenyl)thioureal]. We report preliminary observations with binding FABA (selective for cis, vicdiol structures) to damage sites present on intact nonirradiated and irradiated DNA from C3H10T[sub 1/2] cells. We have observed binding of 42,000 FABA per mouse tetraploid genome (9 billion base pairs) to the putative thymidylic glycol resulting from normal oxidative processes in nonirradiated DNA. Additional binding of FABA to DNA from cells exposed to 100, 300, and 500 rad shows an exponential increase in binding sites of up to 140,000 with 500 rad exposure. This damage reporter may prove useful in characterizing levels of nonovert and overt oxidative damage to DNA.

  7. APOBEC3A damages the cellular genome during DNA replication.

    PubMed

    Green, Abby M; Landry, Sébastien; Budagyan, Konstantin; Avgousti, Daphne C; Shalhout, Sophia; Bhagwat, Ashok S; Weitzman, Matthew D

    2016-01-01

    The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.

  8. APOBEC3A damages the cellular genome during DNA replication

    PubMed Central

    Green, Abby M.; Landry, Sébastien; Budagyan, Konstantin; Avgousti, Daphne C.; Shalhout, Sophia; Bhagwat, Ashok S.; Weitzman, Matthew D.

    2016-01-01

    ABSTRACT The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors. PMID:26918916

  9. Chemical-induced DNA damage and human cancer risk.

    PubMed

    Poirier, Miriam C

    2012-10-01

    For more than 200 years human cancer induction has been known to be associated with a large variety of chemical exposures. Most exposures to chemical carcinogens occur as a result of occupation, pollution in the ambient environment, lifestyle choices, or pharmaceutical use. Scientific investigations have revealed that the majority of cancer causing chemicals, or chemical carcinogens, act through "genotoxic" or DNA damaging mechanisms, which involve covalent binding of the chemical to DNA (DNA adduct formation). Cancer-inducing exposures are typically frequent and/or chronic over years, and the accumulation of DNA damage or DNA adduct formation is considered to be a necessary requirement for tumor induction. Studies in animal models have indicated that the ability to reduce DNA damage will also result in reduction of tumor risk, leading to the hypothesis that individuals having the highest levels of DNA adducts may have an increased cancer risk, compared to individuals with the lowest levels of DNA adducts. Here we have reviewed twelve investigations showing 2- to 9-fold increased Relative Risks (RR) or Odds Ratios (OR) for cancer in (the 25% of) individuals having the highest DNA adduct levels, compared to (the 25% of) matched individuals with the lowest DNA adducts. These studies also provided preliminary evidence that multiple types of DNA adducts combined, or DNA adducts combined with other risk factors (such as infection or inflammation), may be associated with more than 10-fold higher cancer risks (RR = 34-60), compared to those found with a single carcinogen. Taken together the data suggest that a reduction in human DNA adduct level is likely to produce a reduction in human cancer risk.

  10. Spermatogenesis, DNA damage and DNA repair mechanisms in male infertility.

    PubMed

    Gunes, Sezgin; Al-Sadaan, Maha; Agarwal, Ashok

    2015-09-01

    Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development involving mitosis, meiosis and spermiogenesis. Endogenous and exogenous physical, chemical and biological sources modify the genome of spermatozoa. The genomic integrity and stability of the sperm is protected by DNA repair mechanisms. In the male germline cells, DNA repair mechanisms include nucleotide excision repair, base excision repair, DNA mismatch repair, double strand break repair and post-replication repair. Defects in repair mechanisms cause arrest of spermatogenesis and abnormal recombination, ultimately resulting in male infertility. This review focuses on molecular mechanisms of the DNA repair pathways, DNA repair defects and male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  11. Mouse Zygotes Respond to Severe Sperm DNA Damage by Delaying Paternal DNA Replication and Embryonic Development

    PubMed Central

    Gawecka, Joanna E.; Marh, Joel; Ortega, Michael; Yamauchi, Yasuhiro; Ward, Monika A.; Ward, W. Steven

    2013-01-01

    Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development. PMID:23431372

  12. A computational atomistic model of radiation damage to DNA

    NASA Astrophysics Data System (ADS)

    Aydogan, Bulent

    A review of past and current biophysical models of DNA damage reveals that current DNA damage models have become increasingly complex in their attempts to model the full 3D structure of the nucleosome and chromatin fiber. As such, many of the finer details of direct, quasi-direct, and indirect action on DNA become difficult to study in isolation. Also, experimental comparisons that seek to validate these models become increasingly difficult to make. A better approach may be to perform the atomistic modeling of direct, indirect, and quasi-direct effects in total isolation from considerations of the macroscopic conformation of the DNA target. This would permit the highly detailed atomistic modeling to be performed only once in order to produce a database of outcome probabilities that can then be used in radiation chemistry modeling of different and more complex conformations of double-stranded DNA. This work is performed to establish the groundwork to accomplish this goal. A system of Monte Carlo computer codes that model radiation damage to DNA at the atomistic level is developed and used to predict the radiation damage to a 167-bp DNA molecule. A database of the OOH attack outcomes is generated for a 167-bp DNA molecule and used in the prediction of radiation-induced damage to DNA. Do (the dose required to create, on average, one single strand break per 167-bp DNA molecule) is calculated to be 69.9 Gy. There are no experimental study found in the literature that studied small DNA molecules like the one used in this study. Nevertheless, the results from this computational study can be compared to experimental studies preformed with larger DNA molecules such as plasmids when DNA concentrations are scaled. The `concentration scaled D0 (ssb)' values from Klimczak et al. [1993] and Tomita et al. [1998] were approximately 65 and 80 Gy, respectively. These experimental results compare favorably with the computational value of 69.9 Gy calculated in this study. With the

  13. DNA Damage in Chronic Kidney Disease: Evaluation of Clinical Biomarkers

    PubMed Central

    Schupp, Nicole; Stopper, Helga; Heidland, August

    2016-01-01

    Patients with chronic kidney disease (CKD) exhibit an increased cancer risk compared to a healthy control population. To be able to estimate the cancer risk of the patients and to assess the impact of interventional therapies thereon, it is of particular interest to measure the patients' burden of genomic damage. Chromosomal abnormalities, reduced DNA repair, and DNA lesions were found indeed in cells of patients with CKD. Biomarkers for DNA damage measurable in easily accessible cells like peripheral blood lymphocytes are chromosomal aberrations, structural DNA lesions, and oxidatively modified DNA bases. In this review the most common methods quantifying the three parameters mentioned above, the cytokinesis-block micronucleus assay, the comet assay, and the quantification of 8-oxo-7,8-dihydro-2′-deoxyguanosine, are evaluated concerning the feasibility of the analysis and regarding the marker's potential to predict clinical outcomes. PMID:27313827

  14. Connecting the Dots: From DNA Damage and Repair to Aging

    PubMed Central

    Pan, Mei-Ren; Li, Kaiyi; Lin, Shiaw-Yih; Hung, Wen-Chun

    2016-01-01

    Mammalian cells evolve a delicate system, the DNA damage response (DDR) pathway, to monitor genomic integrity and to prevent the damage from both endogenous end exogenous insults. Emerging evidence suggests that aberrant DDR and deficient DNA repair are strongly associated with cancer and aging. Our understanding of the core program of DDR has made tremendous progress in the past two decades. However, the long list of the molecules involved in the DDR and DNA repair continues to grow and the roles of the new “dots” are under intensive investigation. Here, we review the connection between DDR and DNA repair and aging and discuss the potential mechanisms by which deficient DNA repair triggers systemic effects to promote physiological or pathological aging. PMID:27164092

  15. Functions of PARylation in DNA Damage Repair Pathways.

    PubMed

    Wei, Huiting; Yu, Xiaochun

    2016-06-01

    Protein poly ADP-ribosylation (PARylation) is a widespread post-translational modification at DNA lesions, which is catalyzed by poly(ADP-ribose) polymerases (PARPs). This modification regulates a number of biological processes including chromatin reorganization, DNA damage response (DDR), transcriptional regulation, apoptosis, and mitosis. PARP1, functioning as a DNA damage sensor, can be activated by DNA lesions, forming PAR chains that serve as a docking platform for DNA repair factors with high biochemical complexity. Here, we highlight molecular insights into PARylation recognition, the expanding role of PARylation in DDR pathways, and the functional interaction between PARylation and ubiquitination, which will offer us a better understanding of the biological roles of this unique post-translational modification. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  16. NBS1 and multiple regulations of DNA damage response

    PubMed Central

    Komatsu, Kenshi

    2016-01-01

    DNA damage response is finely tuned, with several pathways including those for DNA repair, chromatin remodeling and cell cycle checkpoint, although most studies to date have focused on single pathways. Genetic diseases characterized by genome instability have provided novel insights into the underlying mechanisms of DNA damage response. NBS1, a protein responsible for the radiation-sensitive autosomal recessive disorder Nijmegen breakage syndrome, is one of the first factors to accumulate at sites of DNA double-strand breaks (DSBs). NBS1 binds to at least five key proteins, including ATM, RPA, MRE11, RAD18 and RNF20, in the conserved regions within a limited span of the C terminus, functioning in the regulation of chromatin remodeling, cell cycle checkpoint and DNA repair in response to DSBs. In this article, we reviewed the functions of these binding proteins and their comprehensive association with NBS1. PMID:27068998

  17. Damaging the Integrated HIV Proviral DNA with TALENs

    PubMed Central

    Strong, Christy L.; Guerra, Horacio P.; Mathew, Kiran R.; Roy, Nervik; Simpson, Lacy R.; Schiller, Martin R.

    2015-01-01

    HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs) to target a highly conserved sequence in the transactivation response element (TAR) of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication. PMID:25946221

  18. Modulation of irinotecan-induced genomic DNA damage by theanine.

    PubMed

    Attia, Sabry

    2012-05-01

    The possible chemoprotective activity of theanine against irinotecan-induced genomic DNA damage towards mouse bone marrow cells was investigated. Chromosomal aberrations, DNA damage, micronuclei formation and mitotic activity were studied in the current study as markers of genomic damage. Oxidative DNA stress markers such as 8-hydroxydeoxyguanosine, lipid peroxidation, reduced and oxidized glutathione levels were assessed as a possible mechanism underlying this amelioration. Theanine was neither genotoxic nor cytotoxic in mice at doses equivalent to 30 or 60 mg/kg for 12 days. Pretreatment of mice with theanine significantly reduced irinotecan-induced genomic damage in the bone marrow cells and these effects were dose dependent. Irinotecan induced marked biochemical alterations characteristic of oxidative DNA stress, including increased 8-hydroxydeoxyguanosine, enhanced lipid peroxidation and reduction in the reduced/oxidized glutathione ratio. Prior administration of theanine ahead of irinotecan challenge ameliorated these oxidative DNA stress markers. Overall, this study provides for the first time that theanine has a protective role in the abatement of irinotecan-induced genomic damage in the bone marrow cells of mice that resides, at least in part, on its ability to modulate the cellular antioxidant levels and consequently protect bone marrow from irinotecan genotoxicity.

  19. PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage.

    PubMed

    Riccio, Amanda A; Cingolani, Gino; Pascal, John M

    2016-02-29

    Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage

    PubMed Central

    Riccio, Amanda A.; Cingolani, Gino; Pascal, John M.

    2016-01-01

    Poly(ADP-ribose) polymerase-2 (PARP-2) is one of three human PARP enzymes that are potently activated during the cellular DNA damage response (DDR). DDR-PARPs detect DNA strand breaks, leading to a dramatic increase in their catalytic production of the posttranslational modification poly(ADP-ribose) (PAR) to facilitate repair. There are limited biochemical and structural insights into the functional domains of PARP-2, which has restricted our understanding of how PARP-2 is specialized toward specific repair pathways. PARP-2 has a modular architecture composed of a C-terminal catalytic domain (CAT), a central Trp-Gly-Arg (WGR) domain and an N-terminal region (NTR). Although the NTR is generally considered the key DNA-binding domain of PARP-2, we report here that all three domains of PARP-2 collectively contribute to interaction with DNA damage. Biophysical, structural and biochemical analyses indicate that the NTR is natively disordered, and is only required for activation on specific types of DNA damage. Interestingly, the NTR is not essential for PARP-2 localization to sites of DNA damage. Rather, the WGR and CAT domains function together to recruit PARP-2 to sites of DNA breaks. Our study differentiates the functions of PARP-2 domains from those of PARP-1, the other major DDR-PARP, and highlights the specialization of the multi-domain architectures of DDR-PARPs. PMID:26704974

  1. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-06

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses.

  2. Involvement of DNA Damage Response Pathways in Hepatocellular Carcinoma

    PubMed Central

    Yang, Sheau-Fang; Wei, Ren-Jie; Shiue, Yow-Ling; Wang, Shen-Nien

    2014-01-01

    Hepatocellular carcinoma (HCC) has been known as one of the most lethal human malignancies, due to the difficulty of early detection, chemoresistance, and radioresistance, and is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. Its development has been closely associated with multiple risk factors, including hepatitis B and C virus infection, alcohol consumption, obesity, and diet contamination. Genetic alterations and genomic instability, probably resulted from unrepaired DNA lesions, are increasingly recognized as a common feature of human HCC. Dysregulation of DNA damage repair and signaling to cell cycle checkpoints, known as the DNA damage response (DDR), is associated with a predisposition to cancer and affects responses to DNA-damaging anticancer therapy. It has been demonstrated that various HCC-associated risk factors are able to promote DNA damages, formation of DNA adducts, and chromosomal aberrations. Hence, alterations in the DDR pathways may accumulate these lesions to trigger hepatocarcinogenesis and also to facilitate advanced HCC progression. This review collects some of the most known information about the link between HCC-associated risk factors and DDR pathways in HCC. Hopefully, the review will remind the researchers and clinicians of further characterizing and validating the roles of these DDR pathways in HCC. PMID:24877058

  3. Repair of damaged DNA in vivo: Final technical report

    SciTech Connect

    Hanawalt, P.C.

    1987-09-01

    This contract was initiated in 1962 with the US Atomic Energy Commission to carry out basic research on the effects of radiation on the process of DNA replication in bacteria. Within the first contract year we discovered repair replication at the same time that Setlow and Carrier discovered pyrimidine dimer excision. These discoveries led to the elucidation of the process of excision-repair, one of the most important mechanisms by which living systems, including humans, respond to structural damage in their genetic material. We improved methodology for distinguishing repair replication from semiconservative replication and instructed others in these techniques. Painter then was the first to demonstrate repair replication in ultraviolet irradiated human cells. He, in turn, instructed James Cleaver who discovered that skin fibroblasts from patients with xeroderma pigmentosum were defective in excision-repair. People with this genetic defect are extremely sensitive to sunlight and they develop carcinomas and melanomas of the skin with high frequency. The existence of this hereditary disease attests to the importance of DNA repair in man. We certainly could not survive in the normal ultraviolet flux from the sun if our DNA were not continuously monitored for damage and repaired. Other hereditary diseases such as ataxia telangiectasia, Cockayne's syndrome, Blooms syndrome and Fanconi's anemia also involve deficiencies in DNA damage processing. The field of DNA repair has developed rapidly as we have learned that most environmental chemical carcinogens as well as radiation produce repairable damage in DNA. 251 refs.

  4. DNA damage repair in breast cancer and its therapeutic implications.

    PubMed

    Ali, Reem; Rakha, Emad A; Madhusudan, Srinivasan; Bryant, Helen E

    2017-02-01

    The DNA damage response (DDR) involves the activation of numerous cellular activities that repair DNA lesions and maintain genomic integrity, and is critical in preventing tumorigenesis. Inherited or acquired mutations in specific genes involved in the DNA damage response, for example the breast cancer susceptibility genes 1/2 (BRCA1/2), phosphatase and tensin homolog (PTEN) and P53 are associated with various subtypes of breast cancer. Such changes can render breast cancer cells particularly sensitive to specific DNA damage response inhibitors, for example BRCA1/2 germline mutated cells are sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. The aims of this review are to discuss specific DNA damage response defects in breast cancer and to present the current stage of development of various DDR inhibitors (namely PARP, ATM/ATR, DNA-PK, PARG, RECQL5, FEN1 and APE1) for breast cancer mono- and combination therapy. Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

  5. DNA Damage: From Chronic Inflammation to Age-Related Deterioration

    PubMed Central

    Ioannidou, Anna; Goulielmaki, Evi; Garinis, George A.

    2016-01-01

    To lessen the “wear and tear” of existence, cells have evolved mechanisms that continuously sense DNA lesions, repair DNA damage and restore the compromised genome back to its native form. Besides genome maintenance pathways, multicellular organisms may also employ adaptive and innate immune mechanisms to guard themselves against bacteria or viruses. Recent evidence points to reciprocal interactions between DNA repair, DNA damage responses and aspects of immunity; both self-maintenance and defense responses share a battery of common players and signaling pathways aimed at safeguarding our bodily functions over time. In the short-term, this functional interplay would allow injured cells to restore damaged DNA templates or communicate their compromised state to the microenvironment. In the long-term, however, it may result in the (premature) onset of age-related degeneration, including cancer. Here, we discuss the beneficial and unrewarding outcomes of DNA damage-driven inflammation in the context of tissue-specific pathology and disease progression. PMID:27826317

  6. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  7. SOS processing of unique oxidative DNA damages in Escherichia coli.

    PubMed

    Laspia, M F; Wallace, S S

    1989-05-05

    phi X174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli. All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases. When phi X DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts. Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation. In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts. These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent. Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuC lexA recA-independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.

  8. Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure.

    PubMed

    Qiu, J; Hales, B F; Robaire, B

    1995-12-01

    Treatment of male rats with low dosages of cyclophosphamide causes a dramatic increase in early embryo death among their progeny without significantly affecting the general health of the male. It is hypothesized that cyclophosphamide exerts its effects by targeting specific components of spermatozoal nuclei. The purpose of the present studies was to investigate the effects of chronic cyclophosphamide treatment on spermatozoal DNA. Two approaches were pursued. The first was to determine total DNA damage by using the alkaline elution method. The second was to study spermatozoal DNA template function by using an in vitro DNA synthesis system. Adult male rats were treated with saline or cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected and subjected to alkaline elution using DNA-DNA dot hybridization to quantify the fractionated DNA. One week of treatment with cyclophosphamide caused DNA single strand breaks that could be detected only in the presence of proteinase K in the lysis solution; no DNA cross-links were observed in the animals that received 1-wk drug treatment. In contrast, 6 wk of treatment with cyclophosphamide induced a significant increase in both DNA single strand breaks and cross-links in spermatozoal nuclei; the cross-links were attributable primarily to DNA-DNA linkages. The availability of spermatozoal DNA for template function was not affected by 1 wk of treatment with cyclophosphamide but was markedly affected after 6 wk of treatment with this drug. It is proposed that during chromatin transition processes the male genome may be in an open dynamic state with many exposed sites that are vulnerable to alkylating agents. Since there is no DNA repair during spermiogenesis, damage to the genome by alkylation at this stage may be cumulative, resulting in the production of dysfunctional germ cells.

  9. Parvovirus diversity and DNA damage responses.

    PubMed

    Cotmore, Susan F; Tattersall, Peter

    2013-02-01

    Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral "rolling hairpin" DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ~260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell's entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment.

  10. DNA Damage and L1 Retrotransposition

    PubMed Central

    Farkash, Evan A.; Prak, Eline T. Luning

    2006-01-01

    Barbara McClintock was the first to suggest that transposons are a source of genome instability and that genotoxic stress assisted in their mobilization. The generation of double-stranded DNA breaks (DSBs) is a severe form of genotoxic stress that threatens the integrity of the genome, activates cell cycle checkpoints, and, in some cases, causes cell death. Applying McClintock's stress hypothesis to humans, are L1 retrotransposons, the most active autonomous mobile elements in the modern day human genome, mobilized by DSBs? Here, evidence that transposable elements, particularly retrotransposons, are mobilized by genotoxic stress is reviewed. In the setting of DSB formation, L1 mobility may be affected by changes in the substrate for L1 integration, the DNA repair machinery, or the L1 element itself. The review concludes with a discussion of the potential consequences of L1 mobilization in the setting of genotoxic stress. PMID:16877815

  11. Activation of ATM by DNA Damaging Agents

    DTIC Science & Technology

    2005-09-01

    whereas the related bioflavonoid quercetin induced phosphoryl- for a further 60 or 120 min, and extracts were then prepared ation at these sites in a...cell cycle in response to ionizing radiation- induced DNA double-strand breaks (DSBs). Carriers ofA TM mutations are at increased risk for breast...cancer. Since many anti-tumor chemotherapeutics used in breast cancer treatment have the capacity to induce DSBs, I have investigated the requirement for

  12. Looking for Waldo: A Potential Thermodynamic Signature to DNA Damage

    PubMed Central

    2015-01-01

    Conspectus DNA in its simplest form is an ensemble of nucleic acids, water, and ions, and the conformation of DNA is dependent on the relative proportions of all three components. When DNA is covalently damaged by endogenous or exogenous reactive species, including those produced by some anticancer drugs, the ensemble undergoes localized changes that affect nucleic acid structure, thermodynamic stability, and the qualitative and quantative arrangement of associated cations and water molecules. Fortunately, the biological effects of low levels of DNA damage are successfully mitigated by a large number of proteins that efficiently recognize and repair DNA damage in the midst of a vast excess of canonical DNA. In this Account, we explore the impact of DNA modifications on the high resolution and dynamic structure of DNA, DNA stability, and the uptake of ions and water and explore how these changes may be sensed by proteins whose function is to initially locate DNA lesions. We discuss modifications on the nucleobases that are located in the major and minor grooves of DNA and include lesions that are observed in vivo, including oxidized bases, as well as some synthetic nucleobases that allow us to probe how the location and nature of different substituents affect the thermodynamics and structure of the DNA ensemble. It is demonstrated that disruption of a cation binding site in the major groove by modification of the N7-position on the purines, which is the major site for DNA alkylation, is enthalpically destabilizing. Accordingly, tethering a cationic charge in the major groove is enthalpically stabilizing. The combined structural and thermodynamic studies provide a detailed picture of how different DNA lesions affect the dynamics of DNA and how modified bases interact with their environment. Our work supports the hypothesis that there is a “thermodynamic signature” to DNA lesions that can be exploited in the initial search that requires differentiation between

  13. Looking for Waldo: a potential thermodynamic signature to DNA damage.

    PubMed

    Gold, Barry; Stone, Michael P; Marky, Luis A

    2014-04-15

    DNA in its simplest form is an ensemble of nucleic acids, water, and ions, and the conformation of DNA is dependent on the relative proportions of all three components. When DNA is covalently damaged by endogenous or exogenous reactive species, including those produced by some anticancer drugs, the ensemble undergoes localized changes that affect nucleic acid structure, thermodynamic stability, and the qualitative and quantative arrangement of associated cations and water molecules. Fortunately, the biological effects of low levels of DNA damage are successfully mitigated by a large number of proteins that efficiently recognize and repair DNA damage in the midst of a vast excess of canonical DNA. In this Account, we explore the impact of DNA modifications on the high resolution and dynamic structure of DNA, DNA stability, and the uptake of ions and water and explore how these changes may be sensed by proteins whose function is to initially locate DNA lesions. We discuss modifications on the nucleobases that are located in the major and minor grooves of DNA and include lesions that are observed in vivo, including oxidized bases, as well as some synthetic nucleobases that allow us to probe how the location and nature of different substituents affect the thermodynamics and structure of the DNA ensemble. It is demonstrated that disruption of a cation binding site in the major groove by modification of the N7-position on the purines, which is the major site for DNA alkylation, is enthalpically destabilizing. Accordingly, tethering a cationic charge in the major groove is enthalpically stabilizing. The combined structural and thermodynamic studies provide a detailed picture of how different DNA lesions affect the dynamics of DNA and how modified bases interact with their environment. Our work supports the hypothesis that there is a "thermodynamic signature" to DNA lesions that can be exploited in the initial search that requires differentiation between canonical DNA and

  14. Cellular effects of UVA: DNA damages

    SciTech Connect

    Peak, M.J.; Peak, J.G.

    1988-01-01

    Ultraviolet radiation between 320 nm and visible light (UVA) is a major component of both solar radiation and suntan lamps, which are being increasingly used in tanning booths. UVA has generally been considered innocuous, partially because DNA does not absorb appreciably in this region, but UVB radiation (290-320 nm) has widely been considered to be the major etiological factor in human skin carcinogenesis caused by solar uv radiation, largely because DNA absorbs photons of UVB, which is known to produce thymine photoproducts (cyclobutane dimers and adducts). It is true that patients with xeroderma pigmentosum are particularly prone to solar-uv-induced skin cancer, and cells derived from these people have been shown to lack ability to repair pyrimidine photoproducts by excision, evidence that pyrimidine photoproducts might play a role in carcinogenesis in certain specialized situations. Normal cells have the ability to repair these UVB-induced lesions. However, UVA is considerably more penetrating and more abundant than UVB, and others have performed a spectral analysis that claimed that 20-60% (depending upon the solar zenith angle) of the toxic biological effects of solar radiation can be attributed to UVA. The fact that UVA radiations are mutagenic provides motivation for studying DNA changes that might be effected by this region of the electromagnetic spectrum.

  15. Lipid peroxidation-DNA damage by malondialdehyde.

    PubMed

    Marnett, L J

    1999-03-08

    Malondialdehyde is a naturally occurring product of lipid peroxidation and prostaglandin biosynthesis that is mutagenic and carcinogenic. It reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine. The major adduct to DNA is a pyrimidopurinone called M1G. Site-specific mutagenesis experiments indicate that M1G is mutagenic in bacteria and is repaired by the nucleotide excision repair pathway. M1G has been detected in liver, white blood cells, pancreas, and breast from healthy human beings at levels ranging from 1-120 per 108 nucleotides. Several different assays for M1G have been described that are based on mass spectrometry, 32P-postlabeling, or immunochemical techniques. Each technique offers advantages and disadvantages based on a combination of sensitivity and specificity. Application of each of these techniques to the analysis of M1G is reviewed and future needs for improvements are identified. M1G appears to be a major endogenous DNA adduct in human beings that may contribute significantly to cancer linked to lifestyle and dietary factors. High throughput methods for its detection and quantitation will be extremely useful for screening large populations. Copyright 1999 Elsevier Science B.V.

  16. E2F-7 couples DNA damage-dependent transcription with the DNA repair process.

    PubMed

    Zalmas, Lykourgos-Panagiotis; Coutts, Amanda S; Helleday, Thomas; La Thangue, Nicholas B

    2013-09-15

    The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.

  17. Utilization of salmon milt DNA against UV damage.

    PubMed

    Sasaki, Yoshiharu; Miyoshi, Daisuke; Sugimoto, Naoki

    2010-04-01

    We examined the effect of ultraviolet (UV) irradiation on the UV spectra and radical scavenging activity of DNA strands and found that the absorption spectra of salmon milt DNA was extended up to about 350 nm after ultraviolet C (UVC, 100-280 nm) irradiation with 300 kJ/m(2). The UV B (UVB, 280-315 nm) protection ability of UVC-irradiated salmon milt DNA for a single-stranded target DNA (19-mer) was further studied. The percentage of damaged target DNA after 50 kJ/m(2) of UVB irradiation in the presence of UVC-irradiated salmon milt DNA, UVC-unirradiated salmon milt DNA, and 2-phenylbenzimidazole sulfonic acid was estimated to be 24.6%, 27.0%, and 18.9%, respectively. Moreover, the ultraviolet A (UVA, 315-400 nm)/UVB ratio and critical wavelength of natural (UVC-unirradiated) salmon milt DNA were estimated to be 0.13 and 313 nm, respectively, whereas those of the UVC-irradiated salmon milt DNA were 0.34 and 375 nm, respectively. Interestingly, the value of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity in UVC-irradiated salmon milt DNA was about five times higher than that of UVC-unirradiated salmon milt DNA. These results indicate that the UVC-irradiated salmon milt DNA could be useful as a protector against a wide range of UV light from UVC approximately UVA.

  18. Oxidant-induced DNA damage of target cells.

    PubMed Central

    Schraufstätter, I; Hyslop, P A; Jackson, J H; Cochrane, C G

    1988-01-01

    In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks. PMID:2843565

  19. DNA damage in storage cells of anhydrobiotic tardigrades.

    PubMed

    Neumann, Simon; Reuner, Andy; Brümmer, Franz; Schill, Ralph O

    2009-08-01

    In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 +/- 1.98% DNA in tail, compared to 0.44 +/- 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 +/- 6.41% of DNA was found in the comet tail. After ten months, 23.66 +/- 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.

  20. Detecting ATM-dependent chromatin modification in DNA damage response.

    PubMed

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lopa; Hunt, Clayton; Pandita, Tej K

    2015-01-01

    Loss of function or mutation of the ataxia-telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage.

  1. Detecting ATM-Dependent Chromatin Modification in DNA Damage Response

    PubMed Central

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lope; Hunt, Clayton; Pandita, Tej K.

    2015-01-01

    Loss of function or mutation of the ataxia–telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage. PMID:25827888

  2. Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

    PubMed Central

    Juvekar, Ashish; Hu, Hai; Yadegarynia, Sina; Lyssiotis, Costas A.; Ullas, Soumya; Lien, Evan C.; Bellinger, Gary; Son, Jaekyoung; Hok, Rosanna C.; Seth, Pankaj; Daly, Michele B.; Kim, Baek; Scully, Ralph; Asara, John M.; Cantley, Lewis C.; Wulf, Gerburg M.

    2016-01-01

    We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors. PMID:27402769

  3. Multiomic Analysis of the UV-Induced DNA Damage Response.

    PubMed

    Boeing, Stefan; Williamson, Laura; Encheva, Vesela; Gori, Ilaria; Saunders, Rebecca E; Instrell, Rachael; Aygün, Ozan; Rodriguez-Martinez, Marta; Weems, Juston C; Kelly, Gavin P; Conaway, Joan W; Conaway, Ronald C; Stewart, Aengus; Howell, Michael; Snijders, Ambrosius P; Svejstrup, Jesper Q

    2016-05-11

    In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response.

  4. Mitochondrial and nuclear DNA damage induced by 5-aminolevulinic acid.

    PubMed

    Onuki, Janice; Chen, Yiming; Teixeira, Priscila C; Schumacher, Robert I; Medeiros, Marisa H G; Van Houten, Bennett; Di Mascio, Paolo

    2004-12-15

    5-Aminolevulinic acid (ALA) is a heme precursor accumulated in plasma and in organs in acute intermittent porphyria (AIP), a disease associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma (HCC). Liver biopsies of AIP patients showed odd-shaped mitochondria and autophagic vacuoles containing well-preserved mitochondria. ALA yields reactive oxygen species upon metal-catalyzed oxidation and causes in vivo and in vitro impairment of rat liver mitochondria and DNA damage. Using a quantitative polymerase chain reaction assay, we demonstrated that ALA induces a dose-dependent damage in nuclear and mitochondrial DNA in human SVNF fibroblasts and rat PC12 cells. CHO cells treated with ALA also show nuclear DNA damage and human HepG2 cells entered in apoptosis and necrosis induced by ALA and its dimerization product, DHPY. The present data provide additional information on the genotoxicity of ALA, reinforcing the hypothesis that it may be involved in the development of HCC in AIP patients.

  5. The interplay between inflammation, oxidative stress, DNA damage, DNA repair and mitochondrial dysfunction in depression.

    PubMed

    Czarny, Piotr; Wigner, Paulina; Galecki, Piotr; Sliwinski, Tomasz

    2017-06-29

    A growing body of evidence suggests that inflammation, mitochondrial dysfunction and oxidant-antioxidant imbalance may play a significant role in the development and progression of depression. Elevated levels of reactive oxygen and nitrogen species - a result of oxidant-antioxidant imbalance - may lead to increased damage of biomolecules, including DNA. This was confirmed in depressed patients in a research study conducted by our team and other scientists. 8-oxoguanine - a marker of oxidative DNA damage - was found in the patients' lymphocytes, urine and serum. These results were confirmed using a comet assay on lymphocytes. Furthermore, it was shown that the patients' cells repaired peroxide-induced DNA damage less efficiently than controls' cells and that some single nucleotide polymorphisms (SNP) of the genes involved in oxidative DNA damage repair may modulate the risk of depression. Lastly, less efficient DNA damage repair observed in the patients can be, at least partly, attributed to the presence of specific SNP variants, as it was revealed through a genotype-phenotype analysis. In conclusion, the available literature shows that both oxidative stress and less efficient DNA damage repair may lead to increased DNA damage in depressed patients. A similar mechanism may result in mitochondrial dysfunction, which is observed in depression. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Distribution Patterns of Postmortem Damage in Human Mitochondrial DNA

    PubMed Central

    Gilbert, M. Thomas P.; Willerslev, Eske; Hansen, Anders J.; Barnes, Ian; Rudbeck, Lars; Lynnerup, Niels; Cooper, Alan

    2003-01-01

    The distribution of postmortem damage in mitochondrial DNA retrieved from 37 ancient human DNA samples was analyzed by cloning and was compared with a selection of published animal data. A relative rate of damage (ρv) was calculated for nucleotide positions within the human hypervariable region 1 (HVR1) and cytochrome oxidase subunit III genes. A comparison of damaged sites within and between the regions reveals that damage hotspots exist and that, in the HVR1, these correlate with sites known to have high in vivo mutation rates. Conversely, HVR1 subregions with known structural function, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed “low-diversity 1,” through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions was significantly lower than in the HVR1, and, although hotspots were noted, these did not correlate with codon position. Finally, a simple method for the identification of incorrect archaeological haplogroup designations is introduced, on the basis of the observed spectrum of postmortem damage. PMID:12489041

  7. Analysis of pesticide exposure and DNA damage in immigrant farmworkers.

    PubMed

    McCauley, Linda A; Lasarev, Michael; Muniz, Juan; Nazar Stewart, Valle; Kisby, Glen

    2008-01-01

    In the last decade, the Comet assay has been used increasingly in studies of workers potentially exposed to genotoxic substances in the workplace or environment. Significant increases in DNA damage measured with the Comet assay has been reported in lymphocytes of agricultural workers; however, less intrusive means of biomonitoring are needed in epidemiological investigations. This study was designed to use the Comet assay to describe the association of markers of DNA damage in oral leukocytes with biomarkers of pesticide exposure in 134 farmworkers working in berry crops in Oregon compared to control populations. The authors also examined the extent of DNA damage in young workers compared to adults and the effect of work histories, lifestyle factors, and diet on markers of DNA damage. Urinary levels of organophosphate pesticides were low at the time of sampling; however, mean levels of the Captan metabolite tetrahydrophthalimide (THPI) were found to be shifted significantly higher in the farmworkers (0.14 microg/ml) compared to controls (0.078 microg/ml) (one-sided p value=.01). Likewise, the combined molar equivalent of all dialkylphosphate metabolites was marginally higher in farmworkers (p value=.05). The mean tail intensity was significantly greater for agricultural workers compared to controls (one-sided p value<.001), indicating more DNA damage in the oral leukocytes. On average, the mean tail intensity was 10.9 units greater for agricultural workers (95% CI: 6-16 units greater). Tail moment was also significantly greater for agricultural workers compared to nonagricultural workers (one-sided p value<.001). No Comet parameter was significantly associated with years spent working in agriculture, age, sex, body mass index, diet, and alcohol or tobacco use. The results of this study demonstrate the feasibility for using the Comet assay in biomonitoring studies of farmworkers. Additional studies are needed to examine the effects of different pesticide types on

  8. Can graphene quantum dots cause DNA damage in cells?

    NASA Astrophysics Data System (ADS)

    Wang, Dan; Zhu, Lin; Chen, Jian-Feng; Dai, Liming

    2015-05-01

    Graphene quantum dots (GQDs) have attracted tremendous attention for biological applications. We report the first study on cytotoxicity and genotoxicity of GQDs to fibroblast cell lines (NIH-3T3 cells). The NIH-3T3 cells treated with GQDs at dosages over 50 μg mL-1 showed no significant cytotoxicity. However, the GQD-treated NIH-3T3 cells exhibited an increased expression of proteins (p53, Rad 51, and OGG1) related to DNA damage compared with untreated cells, indicating the DNA damage caused by GQDs. The GQD-induced release of reactive oxygen species (ROS) was demonstrated to be responsible for the observed DNA damage. These findings should have important implications for future applications of GQDs in biological systems.Graphene quantum dots (GQDs) have attracted tremendous attention for biological applications. We report the first study on cytotoxicity and genotoxicity of GQDs to fibroblast cell lines (NIH-3T3 cells). The NIH-3T3 cells treated with GQDs at dosages over 50 μg mL-1 showed no significant cytotoxicity. However, the GQD-treated NIH-3T3 cells exhibited an increased expression of proteins (p53, Rad 51, and OGG1) related to DNA damage compared with untreated cells, indicating the DNA damage caused by GQDs. The GQD-induced release of reactive oxygen species (ROS) was demonstrated to be responsible for the observed DNA damage. These findings should have important implications for future applications of GQDs in biological systems. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01734c

  9. DNA damage and oxidative status in PFAPA syndrome.

    PubMed

    Tuğrul, Selahattin; Doğan, Remzi; Kocyigit, Abdurrahim; Torun, Emel; Senturk, Erol; Ozturan, Orhan

    2015-10-01

    PFAPA syndrome is a clinical entity of unknown etiology which presents with periodic episodes of fever, aphthous stomatitis, tonsillitis or pharyngitis, and cervical adenitis. In this study we investigated DNA damage and the oxidative stress parameters in patients diagnosed with PFAPA, to elucidate the underlying pathophysiological mechanism of this syndrome. Thirty-one patients diagnosed with PFAPA (Group 1), 22 patients diagnosed with normal tonsillitis or pharyngitis (Group 2), and 20 healthy volunteers (Group 3) were included in our study. Heparinized peripheral blood samples were drawn from all patients and volunteers. DNA damage was assessed by single cell alkaline electrophoresis assay in peripheral mononuclear leukocytes. Plasma levels of total antioxidant status (TAS) and total oxidative status (TOS) were determined by using a novel automated measurement method, and oxidative stress index (OSI) was calculated. DNA damage in the mononuclear leukocytes of Group 1 was significantly higher than that of Group 2 and Group 3. The oxidative stress parameters revealed that the TOS and OSI values of Group 1 were significantly higher than those of Group 2 and Group 3. TAS values of Group 1 were significantly lower than those of Group 2 and Group 3. Correlation analysis of Group 1 demonstrated a significant correlation between TOS, one of the oxidative stress parameters, and DNA damage. Correlations between DNA damage and C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values were also significant. Our study indicated that both the inflammatory and the oxidative stress parameters were significantly increased in patients with PFAPA syndrome, accompanied by a significant positive correlation between DNA damage and oxidative stress. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. BACH2: a marker of DNA damage and ageing.

    PubMed

    Uittenboogaard, L M; Payan-Gomez, C; Pothof, J; van Ijcken, W; Mastroberardino, P G; van der Pluijm, I; Hoeijmakers, J H J; Tresini, M

    2013-11-01

    DNA damage and ageing share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. "Omics" technologies have permitted large-scale parallel measurements covering global cellular constituents and aided the identification of specific response pathways that change during ageing and after DNA damage. We have set out to identify genes with highly conserved response patterns through meta-analysis of mRNA expression datasets collected during natural ageing and accelerated ageing caused by a Transcription-Coupled Nucleotide Excision Repair (TC-NER) defect in a diverse set of organs and tissues in mice, and from in vitro UV-induced DNA damage in a variety of murine cells. The identified set of genes that show similar expression patterns in response to organ ageing (accelerated and normal), and endogenously and exogenously induced DNA damage, consists of genes involved in anti-oxidant systems and includes the transcription factor Bach2 as one of the most consistent markers. BACH2 was originally identified as a partner of the small Maf proteins and antagonist of the NRF2 anti-oxidant defence pathway and has been implicated in B-cell differentiation and immune system homeostasis. Although BACH2 has never before been associated with UV-induced damage or ageing, it shows a strong downregulation in both conditions. We have characterized the dynamics of Bach2 expression in response to DNA damage and show that it is a highly sensitive responder to transcription-blocking DNA lesions. Gene expression profiling using Affymetrix microarray analysis after siRNA-mediated silencing of Bach2 identified cell cycle and transcription regulation as the most significantly altered processes consistent with a function as transcription factor affecting proliferation.

  11. BACH2: a Marker of DNA Damage and Aging

    PubMed Central

    Uittenboogaard, L.M.; Payan-Gomez, C.; Pothof, J.; van IJcken, W.; Mastroberardino, PG; van der Pluijm; Hoeijmakers, J.H.J.; Tresini, M.

    2013-01-01

    DNA damage and aging share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. “Omics” technologies have permitted large-scale parallel measurements covering global cellular constituents and aided the identification of specific response pathways that change during aging and after DNA damage. We have set out to identify genes with highly conserved response patterns through meta-analysis of mRNA expression datasets collected during natural aging and accelerated aging caused by a Transcription-Coupled Nucleotide Excision Repair (TC-NER) defect in a diverse set of organs and tissues in mice, and from in-vitro UV-induced DNA damage in a variety of murine cells. The identified set of genes that show similar expression patterns in response to organ aging (accelerated and normal), and endogenously and exogenously induced DNA damage, consists of genes involved in anti-oxidant systems and includes the transcription factor Bach2 as one of the most consistent markers. BACH2 was originally identified as a partner of the small Maf proteins and antagonist of the NRF2 anti-oxidant defence pathway and has been implicated in B-cell differentiation and immune system homeostasis. Although BACH2 has never before been associated with UV-induced damage or aging, it shows a strong downregulation in both conditions. We have characterized the dynamics of Bach2 expression in response to DNA damage and show that it is a highly sensitive responder to transcription-blocking DNA lesions. Gene expression profiling using Affymetrix microarray analysis after siRNA-mediated silencing of Bach2 identified cell cycle and transcription regulation as the most significantly altered processes consistent with a function as transcription factor affecting proliferation. PMID:24075570

  12. 5-Lipoxygenase-mediated endogenous DNA damage.

    PubMed

    Jian, Wenying; Lee, Seon Hwa; Williams, Michelle V; Blair, Ian A

    2009-06-19

    Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 +/- 0.01 to 45.50 +/- 4.05 pmol/10(7) cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2'-deoxyguanosine (HepsilondGuo) from 2.41 +/- 0.35 to 6.31 +/- 0.73 adducts/10(7) normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HepsilondGuo DNA-adduct in CESS cells.

  13. Mitochondrial DNA Damage in Iron Overload*S⃞

    PubMed Central

    Gao, Xueshan; Campian, Jian Li; Qian, Mingwei; Sun, Xiao-Feng; Eaton, John W.

    2009-01-01

    Chronic iron overload has slow and insidious effects on heart, liver, and other organs. Because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological “memory.” We hypothesized that cumulative iron-catalyzed oxidant damage to mtDNA might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. Real time PCR was used to examine the “intactness” of mttDNA in cultured H9c2 rat cardiac myocytes. After 3–5 days exposure to high iron, these cells exhibited damage to mtDNA reflected by diminished amounts of near full-length 15.9-kb PCR product with no change in the amounts of a 16.1-kb product from a nuclear gene. With the loss of intact mtDNA, cellular respiration declined and mRNAs for three electron transport chain subunits and 16 S rRNA encoded by mtDNA decreased, whereas no decrements were found in four subunits encoded by nuclear DNA. To examine the importance of the interactions of iron with metabolically generated reactive oxygen species, we compared the toxic effects of iron in wild-type and rhoo cells. In wild-type cells, elevated iron caused increased production of reactive oxygen species, cytostasis, and cell death, whereas the rhoo cells were unaffected. We conclude that long-term damage to cells and organs in iron-overload disorders involves interactions between iron and mitochondrial reactive oxygen species resulting in cumulative damage to mtDNA, impaired synthesis of respiratory chain subunits, and respiratory dysfunction. PMID:19095657

  14. Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

    PubMed

    Doudican, Nicole A; Song, Binwei; Shadel, Gerald S; Doetsch, Paul W

    2005-06-01

    Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

  15. Repair Machinery for Radiation-Induced DNA Damage

    DTIC Science & Technology

    2000-07-01

    significant defect in the repair of certain DNA damages, but of which damages needs to be determined. We have selected Chinese Hamster Ovary ( CHO ) as...chromosome (BAC) genomic fragment, which we isolated from a CHO BAC library, revealed that APE1 exists as a single copy gene in AA8 (see Appendix, Figure... cells , we first determined the APE1 gene copy number in the CHO AA8 cell line. Fluorescence in situ hybridization with an APE1 bacterial artificial

  16. Phase resetting of the mammalian circadian clock by DNA damage.

    PubMed

    Oklejewicz, Małgorzata; Destici, Eugin; Tamanini, Filippo; Hut, Roelof A; Janssens, Roel; van der Horst, Gijsbertus T J

    2008-02-26

    To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

  17. DNA damage and repair in human skin in situ

    SciTech Connect

    Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

    1987-01-01

    Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs.

  18. DNA damage response during mouse oocyte maturation

    PubMed Central

    Mayer, Alexandra; Baran, Vladimir; Sakakibara, Yogo; Brzakova, Adela; Ferencova, Ivana; Motlik, Jan; Kitajima, Tomoya S.; Schultz, Richard M.; Solc, Petr

    2016-01-01

    ABSTRACT Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation. PMID:26745237

  19. Nuclear DNA damage signalling to mitochondria in ageing

    PubMed Central

    Fang, Evandro Fei; Scheibye-Knudsen, Morten; Chua, Katrin F.; Mattson, Mark P.; Croteau, Deborah L.; Bohr, Vilhelm A.

    2016-01-01

    Mitochondrial dysfunction is a hallmark of ageing, and mitochondrial maintenance may lead to increased healthspan. Emerging evidence suggests a crucial role for signalling from the nucleus to mitochondria (NM signalling) in regulating mitochondrial function and ageing. An important initiator of NM signalling is nuclear DNA damage, which accumulates with age and may contribute to the development of age-associated diseases. DNA damage-dependent NM signalling constitutes a network that includes nuclear sirtuins and controls genomic stability and mitochondrial integrity. Pharmacological modulation of NM signalling is a promising novel approach for the prevention and treatment of age-associated diseases. PMID:26956196

  20. Linking abnormal mitosis to the acquisition of DNA damage

    PubMed Central

    Pellman, David

    2012-01-01

    Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis. PMID:23229895

  1. Mechanisms Used by Plants to Cope with DNA Damage.

    PubMed

    Hu, Zhubing; Cools, Toon; De Veylder, Lieven

    2016-04-29

    Because the genome stores all genetic information required for growth and development, it is of pivotal importance to maintain DNA integrity, especially during cell division, when the genome is prone to replication errors and damage. Although over the last two decades it has become evident that the basic cell cycle toolbox of plants shares several similarities with those of fungi and mammals, plants appear to have evolved a set of distinct checkpoint regulators in response to different types of DNA stress. This might be a consequence of plants' sessile lifestyle, which exposes them to a set of unique DNA damage-inducing conditions. In this review, we highlight the types of DNA stress that plants typically experience and describe the plant-specific molecular mechanisms that control cell division in response to these stresses.

  2. Voltammetric Behavior of o-Nitrophenol and Damage to DNA

    PubMed Central

    Zhang, Da-Peng; Wu, Wei-Li; Long, Hai-Yan; Liu, Yun-Chun; Yang, Zhou-Sheng

    2008-01-01

    The electrochemical behavior of o-nitrophenol was studied in detail with a glassy carbon electrode (GCE). The dependence of peak potential on pH indicated that equivalent electrons and protons were involved in the process of o-nitrophenol reduction. The interaction of o-nitrophenol with calf thymus DNA was investigated by adding DNA to the o-nitrophenol solution and by immobilizing DNA on GCE, respectively. The peak current decrement and peak potential shift in presence of DNA indicated that o-nitrophenol could interact with DNA. The result was demonstrated that the in situ DNA damage was detected by differential pulse voltammetry after the o-nitrophenol was electrochemically reduced. PMID:19325751

  3. Glycosylases utilize ``stop and go'' motion to locate DNA damage

    NASA Astrophysics Data System (ADS)

    Nelson, Shane

    2015-03-01

    Oxidative damage to DNA results in alterations that are mutagenic or even cytotoxic. Base excision repair is a mechanism that functions to identify and correct these lesions, and is present in organisms ranging from bacteria to humans. DNA glycosylases are the first enzymes in this pathway and function to locate and remove oxidatively damaged bases, and do so utilizing only thermal energy. However, the question remains of how these enzymes locate and recognize a damaged base among millions of undamaged bases. Utilizing fluorescence video microscopy with high spatial and temporal resolution, we have observed a number of different fluorescently labeled glycosylases (including bacterial FPG, NEI, and NTH as well as mammalian MutyH and OGG). These enzymes diffuse along DNA tightropes at approximately 0.01 +/- 0.005 μm2/s with binding lifetimes ranging from one second to several minutes. Chemically induced damage to the DNA substrate causes a ~ 50% reduction in diffusion coefficients and a ~ 400% increase in binding lifetimes, while mutation of the key ``wedge residue'' - which has been shown to be responsible for damage detection - results in a 200% increase in the diffusion coefficient. Utilizing a sliding window approach to measure diffusion coefficients within individual trajectories, we observe that distributions of diffusion coefficients are bimodal, consistent with periods of diffusive motion interspersed with immobile periods. Utilizing a unique chemo-mechanical simulation approach, we demonstrate that the motion of these glycosylases can be explained as free diffusion along the helical pitch of the DNA, punctuated with two different types of pauses: 1) rapid, short-lived pauses as the enzyme rapidly probes DNA bases to interrogate for damage and, 2) less frequent, longer lived pauses that reflect the enzyme bound to and catalytically removing a damaged base. These simulations also indicate that the wedge residue is critical for interrogation and recognition of

  4. DNA damage mediated transcription arrest: Step back to go forward.

    PubMed

    Mullenders, Leon

    2015-12-01

    The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Photo-induced DNA damage, DNA repair and cell lethality

    SciTech Connect

    Cool, B.L.

    1982-01-01

    DNA lesion induction and repair was measured in DNA repair proficient and deficient cells after exposures to far-UV, mid-UV, near-UV and visible light and an attempt was made to relate these molecular phenomena to the biological endpoint of cell lethality. Pyrimidine dimer and strand break induction, DNA repair and cell killing were measured after cell exposure to polychromatic but narrow bandwidth light sources with peak emissions at 254, 305, 353, 369, and 445 nm. Pyrimidine dimers were detected using specific endonuclease that nicks DNA adjacent to dimers, while strand breaks were measured using an alkaline unwinding assay. The induction efficiencies of both lesions declined with increasing wavelength; however, the decrease in strand break induction was not as rapid as that of dimer induction. The ratio of strand breaks to dimers following cell exposure to 254 or 369 nm radiation was, respectively, 1.8 x 10/sup -4/ or 0.19. The kinetics of dimer repair as well as the size of repair synthesized patches remained constant with increasing wavelength, indicating a similar repair mechanism for dimers induced by all wavelengths tested. However, consistent with the detected decline in dimer induction with increasing wavelength the proportion of dimer repair to total DNA repair decreased with increasing wavelength. The efficiency of cell killing, determined using chlonagenic survival assays, dropped rapidly, but not as rapidly as that of dimer induction, with increasing wavelength. In addition, dimer repair deficient xeroderma pigmentosum cells became less lethally hypersensitive with increasing wavelength. These data suggest a decline in dimer induced cell lethality and the existence of non-dimer lethal lesions at longer wavelengths.

  6. Diagnosis of Lung Cancer by Fractal Analysis of Damaged DNA

    PubMed Central

    Namazi, Hamidreza; Kiminezhadmalaie, Mona

    2015-01-01

    Cancer starts when cells in a part of the body start to grow out of control. In fact cells become cancer cells because of DNA damage. A DNA walk of a genome represents how the frequency of each nucleotide of a pairing nucleotide couple changes locally. In this research in order to study the cancer genes, DNA walk plots of genomes of patients with lung cancer were generated using a program written in MATLAB language. The data so obtained was checked for fractal property by computing the fractal dimension using a program written in MATLAB. Also, the correlation of damaged DNA was studied using the Hurst exponent measure. We have found that the damaged DNA sequences are exhibiting higher degree of fractality and less correlation compared with normal DNA sequences. So we confirmed this method can be used for early detection of lung cancer. The method introduced in this research not only is useful for diagnosis of lung cancer but also can be applied for detection and growth analysis of different types of cancers. PMID:26539245

  7. Viral Carcinogenesis: Factors Inducing DNA Damage and Virus Integration

    PubMed Central

    Chen, Yan; Williams, Vonetta; Filippova, Maria; Filippov, Valery; Duerksen-Hughes, Penelope

    2014-01-01

    Viruses are the causative agents of 10%–15% of human cancers worldwide. The most common outcome for virus-induced reprogramming is genomic instability, including accumulation of mutations, aberrations and DNA damage. Although each virus has its own specific mechanism for promoting carcinogenesis, the majority of DNA oncogenic viruses encode oncogenes that transform infected cells, frequently by targeting p53 and pRB. In addition, integration of viral DNA into the human genome can also play an important role in promoting tumor development for several viruses, including HBV and HPV. Because viral integration requires the breakage of both the viral and the host DNA, the integration rate is believed to be linked to the levels of DNA damage. DNA damage can be caused by both endogenous and exogenous factors, including inflammation induced by either the virus itself or by co-infections with other agents, environmental agents and other factors. Typically, cancer develops years to decades following the initial infection. A better understanding of virus-mediated carcinogenesis, the networking of pathways involved in transformation and the relevant risk factors, particularly in those cases where tumorigenesis proceeds by way of virus integration, will help to suggest prophylactic and therapeutic strategies to reduce the risk of virus-mediated cancer. PMID:25340830

  8. High glucose induces DNA damage in cultured human endothelial cells.

    PubMed Central

    Lorenzi, M; Montisano, D F; Toledo, S; Barrieux, A

    1986-01-01

    Morphologic and functional abnormalities of vascular endothelium are well recognized in diabetes. In view of our previous finding that high glucose concentrations accelerate death and hamper replication of cultured human endothelial cells, we have investigated in the same model the possibility that exposure to high glucose may result in DNA damage. DNA from human endothelial cells--but not from fibroblasts--exposed to 30 mM glucose for 9-14 d manifested an accelerated rate of unwinding in alkali indicative of an increased number of single strand breaks (P less than 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333 +/- 153 cpm/10(5) cells vs. 88 +/- 42 in control cells, mean +/- SD, P = 0.04), which is indicative of increased DNA repair synthesis. Neither DNA damage nor repair synthesis were increased by medium hypertonicity achieved with 30 mM mannitol. These findings suggest the possibility that, under conditions of high ambient glucose, excess glucose entry in cells that are insulin independent for glucose transport may, directly or indirectly, perturb DNA function. Further, they suggest the possibility that different individual capabilities to repair DNA damage--a process that is under genetic control--may represent a mechanism for different individual susceptibilities to development of diabetic vascular complication. PMID:3944257

  9. DNA repair mechanisms and the bypass of DNA damage in Saccharomyces cerevisiae.

    PubMed

    Boiteux, Serge; Jinks-Robertson, Sue

    2013-04-01

    DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.

  10. DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

    PubMed Central

    Boiteux, Serge; Jinks-Robertson, Sue

    2013-01-01

    DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

  11. No ancient DNA damage in Actinobacteria from the Neanderthal bone.

    PubMed

    Zaremba-Niedźwiedzka, Katarzyna; Andersson, Siv G E

    2013-01-01

    The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized. Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences. Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

  12. No Ancient DNA Damage in Actinobacteria from the Neanderthal Bone

    PubMed Central

    Zaremba-Niedźwiedzka, Katarzyna; Andersson, Siv G. E.

    2013-01-01

    Background The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized. Principal Findings Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences. Conclusions/Significance Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample. PMID:23658776

  13. Role for DNA damage signaling in pulmonary arterial hypertension.

    PubMed

    Meloche, Jolyane; Pflieger, Aude; Vaillancourt, Mylène; Paulin, Roxane; Potus, François; Zervopoulos, Sotirios; Graydon, Colin; Courboulin, Audrey; Breuils-Bonnet, Sandra; Tremblay, Eve; Couture, Christian; Michelakis, Evangelos D; Provencher, Steeve; Bonnet, Sébastien

    2014-02-18

    Pulmonary arterial hypertension (PAH) is associated with sustained inflammation known to promote DNA damage. Despite these unfavorable environmental conditions, PAH pulmonary arterial smooth muscle cells (PASMCs) exhibit, in contrast to healthy PASMCs, a pro-proliferative and anti-apoptotic phenotype, sustained in time by the activation of miR-204, nuclear factor of activated T cells, and hypoxia-inducible factor 1-α. We hypothesized that PAH-PASMCs have increased the activation of poly(ADP-ribose) polymerase-1 (PARP-1), a critical enzyme implicated in DNA repair, allowing proliferation despite the presence of DNA-damaging insults, eventually leading to PAH. Human PAH distal pulmonary arteries and cultured PAH-PASMCs exhibit increased DNA damage markers (53BP1 and γ-H2AX) and an overexpression of PARP-1 (immunoblot and activity assay), in comparison with healthy tissues/cells. Healthy PASMCs treated with a clinically relevant dose of tumor necrosis factor-α harbored a similar phenotype, suggesting that inflammation induces DNA damage and PARP-1 activation in PAH. We also showed that PARP-1 activation accounts for miR-204 downregulation (quantitative reverse transcription polymerase chain reaction) and the subsequent activation of the transcription factors nuclear factor of activated T cells and hypoxia-inducible factor 1-α in PAH-PASMCs, previously shown to be critical for PAH in several models. These effects resulted in PASMC proliferation (Ki67, proliferating cell nuclear antigen, and WST1 assays) and resistance to apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling and Annexin V assays). In vivo, the clinically available PARP inhibitor ABT-888 reversed PAH in 2 experimental rat models (Sugen/hypoxia and monocrotaline). These results show for the first time that the DNA damage/PARP-1 signaling pathway is important for PAH development and provide a new therapeutic target for this deadly disease with high translational potential.

  14. Mechanism of site-specific DNA damage induced by ozone.

    PubMed

    Ito, Kimiko; Inoue, Sumiko; Hiraku, Yusuke; Kawanishi, Shosuke

    2005-08-01

    Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2'-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.

  15. DNA damage under simulated extraterrestrial conditions in bacteriophage T7

    NASA Astrophysics Data System (ADS)

    Fekete, A.; Kovács, G.; Hegedüs, M.; Módos, K.; Rontó, Gy.; Lammer, H.; Panitz, C.

    The experiment ``Phage and uracil response'' (PUR) will be accommodated in the EXPOSE facility of the ISS aiming to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. To achieve this new method was elaborated for the preparation of DNA and nucleoprotein thin films (1). During the EXPOSE Experiment Verification Tests (EVT) the samples were exposed to vacuum (10 -6 Pa), to monochromatic (254 nm) and polychromatic (200-400 nm) UV radiation in air as well in simulated space vacuum. Using neutral density (ND) filters dose-effect curves were performed in order to define the maximum doses tolerated, and we also studied the effect of temperature in vacuum as well as the influence of temperature fluctuations. We obtained substantial evidence that DNA lesions (e.g. strand breaks, DNA-protein cross-links, DNA-DNA cross-links) accumulate throughout exposure. DNA damage was determined by quantitative PCR using 555 bp and 3826 bp fragments of T7 DNA (2) and by neutral and alkaline agarose gel electrophoresis; the structural/chemical effects were analyzed by spectroscopic and microscopical methods. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of the PCR products have been detected indicating the damage of isolated and intraphage DNA. Preliminary results suggest a synergistic action of space vacuum and UV radiation with DNA being the critical target. Fekete et al. J. Luminescence 102-103, 469-475, 2003 Hegedüs et al. Photochem. Photobiol. 78, 213-219, 2003

  16. Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

    PubMed Central

    Quiñones, A; Kaasch, J; Kaasch, M; Messer, W

    1989-01-01

    The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response. Images PMID:2656258

  17. Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

    PubMed

    Quiñones, A; Kaasch, J; Kaasch, M; Messer, W

    1989-02-01

    The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response.

  18. Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

    PubMed Central

    Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  19. Low-dose formaldehyde delays DNA damage recognition and DNA excision repair in human cells.

    PubMed

    Luch, Andreas; Frey, Flurina C Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks.

  20. Viruses and the DNA Damage Response: Activation and Antagonism.

    PubMed

    Luftig, Micah A

    2014-11-01

    Viruses must interact with their hosts in order to replicate; these interactions often provoke the evolutionarily conserved response to DNA damage, known as the DNA damage response (DDR). The DDR can be activated by incoming viral DNA, during the integration of retroviruses, or in response to the aberrant DNA structures generated upon replication of DNA viruses. Furthermore, DNA and RNA viral proteins can induce the DDR by promoting inappropriate S phase entry, by modifying cellular DDR factors directly, or by unintentionally targeting host DNA. The DDR may be antiviral, although viruses often require proximal DDR activation of repair and recombination factors to facilitate replication as well as downstream DDR signaling suppression to ensure cell survival. An unintended consequence of DDR attenuation during infection is the long-term survival and proliferation of precancerous cells. Therefore, the molecular basis for DDR activation and attenuation by viruses remains an important area of study that will likely provide key insights into how viruses have evolved with their hosts.

  1. Evaluation of DNA damage in flight personnel by Comet assay.

    PubMed

    Cavallo, Delia; Tomao, Paola; Marinaccio, Alessandro; Perniconi, Barbara; Setini, Andrea; Palmi, Silvana; Iavicoli, Sergio

    2002-04-26

    There have been some suggestions that air-crew are at a higher-than-normal risk of developing cancer, since they are exposed to potential genotoxic factors. These include cosmic radiations, airborne pollutants such as the combustion products of jet propulsion, ozone, and electromagnetic fields. We used the Comet assay to investigate DNA damage in flight personnel with the aim of assessing potential health hazards in this occupational category. We studied 40 civil air-crew members who had been flying long-haul routes for at least 5 years, and compared them with a homogeneous control group of 40 healthy male ground staff. The Comet assay, or single-cell gel electrophoresis (SCGE), detects DNA single- and double-strand breaks (DSBs) and alkali-labile lesions in individual cells, and is a powerful and sensitive technique for detecting genetic damage induced by different genotoxic agents. Taking into consideration occupational risk and possible confounding factors, this assay showed a small increase, that did not reach statistical significance, of DNA damage in long-haul crew members compared to controls, indicating a lack of evident genotoxic effects. An association, although again not statistically significant, was found between reduced DNA damage and use of protective drugs (antioxidants).

  2. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    EPA Science Inventory

    A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

  3. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  4. Mitochondrial DNA damage induces apoptosis in senescent cells

    PubMed Central

    Laberge, R-M; Adler, D; DeMaria, M; Mechtouf, N; Teachenor, R; Cardin, G B; Desprez, P-Y; Campisi, J; Rodier, F

    2013-01-01

    Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV–HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells. PMID:23868060

  5. Mitochondrial DNA damage induces apoptosis in senescent cells.

    PubMed

    Laberge, R-M; Adler, D; DeMaria, M; Mechtouf, N; Teachenor, R; Cardin, G B; Desprez, P-Y; Campisi, J; Rodier, F

    2013-07-18

    Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV-HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells.

  6. DNA damage, neuronal and glial cell death and neurodegeneration.

    PubMed

    Barzilai, Ari

    2010-11-01

    The DNA damage response (DDR) is a key factor in the maintenance of genome stability. As such, it is a central axis in sustaining cellular homeostasis in a variety of contexts: development, growth, differentiation, and maintenance of the normal life cycle of the cell. It is now clear that diverse mechanisms encompassing cell cycle regulation, repair pathways, many aspects of cellular metabolism, and cell death are inter-linked and act in concert in response to DNA damage. Defects in the DDR in proliferating cells can lead to cancer, while DDR defects in neurons may result in neurodegeneration. Mature neurons are highly differentiated, post-mitotic cells that cannot be replenished after disease or trauma. Their high metabolic activity generates large amounts of reactive oxygen species with DNA damaging capacity. Moreover, their intense transcriptional activity increases the potential for genomic DNA damage. Respectively, neurons have elaborate mechanisms to defend the integrity of their genome, thus ensuring their longevity and functionality in the face of these threats. Over the course of the past two decades, there has been a substantial increase in our understanding of the role of glial cells in supporting the neuronal cell DDR and longevity. This review article focuses on the potential role of the DDR in the etiology and pathogenesis of neurodegenerative diseases, and in addition, it describes various aspects of glial cell functionality in two genomic instability disorders: ataxia telangiectasia (A-T) and Nijmegen breakage syndrome.

  7. Modeling the Study of DNA Damage Responses in Mice

    PubMed Central

    Specks, Julia; Nieto-Soler, Maria; Lopez-Contreras, Andres J; Fernandez-Capetillo, Oscar

    2016-01-01

    Summary Damaged DNA has a profound impact on mammalian health and overall survival. In addition to being the source of mutations that initiate cancer, the accumulation of toxic amounts of DNA damage can cause severe developmental diseases and accelerate ageing. Therefore, understanding how cells respond to DNA damage has become one of the most intense areas of biomedical research in the recent years. However, whereas most mechanistic studies derive from in vitro or in cellulo work, the impact of a given mutation on a living organism is largely unpredictable. For instance, why BRCA1 mutations preferentially lead to breast cancer whereas mutations compromising mismatch repair drive colon cancer is still not understood. In this context, evaluating the specific physiological impact of mutations that compromise genome integrity has become crucial for a better dimensioning of our knowledge. We here describe the various technologies that can be used for modeling mutations in mice, and provide a review of the genes and pathways that have been modeled so far in the context of DNA damage responses. PMID:25636482

  8. SUMO boosts the DNA damage response barrier against cancer.

    PubMed

    Bartek, Jiri; Hodny, Zdenek

    2010-01-19

    Cells exposed to genotoxic insults such as ionizing radiation activate a signaling cascade to repair the damaged DNA. Two recent articles published in Nature show that such genome maintenance requires modifications of tumor suppressor proteins BRCA1 and 53BP1 by the small ubiquitin-like modifier SUMO. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  9. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    EPA Science Inventory

    A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

  10. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  11. MicroRNAs, DNA Damage Response, and Cancer Treatment

    PubMed Central

    He, Mingyang; Zhou, Weiwei; Li, Chuang; Guo, Mingxiong

    2016-01-01

    As a result of various stresses, lesions caused by DNA-damaging agents occur constantly in each cell of the human body. Generally, DNA damage is recognized and repaired by the DNA damage response (DDR) machinery, and the cells survive. When repair fails, the genomic integrity of the cell is disrupted—a hallmark of cancer. In addition, the DDR plays a dual role in cancer development and therapy. Cancer radiotherapy and chemotherapy are designed to eliminate cancer cells by inducing DNA damage, which in turn can promote tumorigenesis. Over the past two decades, an increasing number of microRNAs (miRNAs), small noncoding RNAs, have been identified as participating in the processes regulating tumorigenesis and responses to cancer treatment with radiation therapy or genotoxic chemotherapies, by modulating the DDR. The purpose of this review is to summarize the recent findings on how miRNAs regulate the DDR and discuss the therapeutic functions of miRNAs in cancer in the context of DDR regulation. PMID:27973455

  12. DNA Damage, Fruits and Vegetables and Breast Cancer Prevention

    DTIC Science & Technology

    2001-08-01

    fruits and vegetables per day for a total of 8 weeks on measures of oxidative damage to DNA and lipids is being determined. Urine and blood ... components are being assessed for oxidative endpoints and plasma is being evaluated for biochemical markers of edible plant consumption. Body Approved

  13. The interaction between polynucleotide kinase phosphatase and the DNA repair protein XRCC1 is critical for repair of DNA alkylation damage and stable association at DNA damage sites.

    PubMed

    Della-Maria, Julie; Hegde, Muralidhar L; McNeill, Daniel R; Matsumoto, Yoshihiro; Tsai, Miaw-Sheue; Ellenberger, Tom; Wilson, David M; Mitra, Sankar; Tomkinson, Alan E

    2012-11-09

    XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.

  14. The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites*

    PubMed Central

    Della-Maria, Julie; Hegde, Muralidhar L.; McNeill, Daniel R.; Matsumoto, Yoshihiro; Tsai, Miaw-Sheue; Ellenberger, Tom; Wilson, David M.; Mitra, Sankar; Tomkinson, Alan E.

    2012-01-01

    XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair. PMID:22992732

  15. Epstein-Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage.

    PubMed

    Yang, Jie; Deng, Wen; Hau, Pok M; Liu, Jia; Lau, Victoria M Y; Cheung, Annie L M; Huen, Michael S Y; Tsao, Sai W

    2015-08-01

    Epstein-Barr virus (EBV) infection is closely associated with several human malignancies including nasopharyngeal carcinoma (NPC). The EBV immediate-early protein BZLF1 is the key mediator that switches EBV infection from latent to lytic forms. The lytic form of EBV infection has been implicated in human carcinogenesis but its molecular mechanisms remain unclear. BZLF1 has been shown to be a binding partner of several DNA damage response (DDR) proteins. Its functions in host DDR remain unknown. Thus, we explore the effects of BZLF1 on cellular response to DNA damage in NPC cells. We found that expression of BZLF1 impaired the binding between RNF8 and MDC1 (mediator of DNA damage checkpoint 1), which in turn interfered with the localization of RNF8 and 53BP1 to the DNA damage sites. The RNF8-53BP1 pathway is important for repair of DNA double-strand breaks and DNA damage-induced G2/M checkpoint activation. Our results showed that, by impairing DNA damage repair as well as abrogating G2/M checkpoint, BZLF1 induced genomic instability and rendered cells more sensitive to ionizing radiation. Moreover, the blockage of 53BP1 and RNF8 foci formation was recapitulated in EBV-infected cells. Taken together, our study raises the possibility that, by causing mis-localization of important DDR proteins, BZLF1 may function as a link between lytic EBV infection and impaired DNA damage repair, thus contributing to the carcinogenesis of EBV-associated human epithelial malignancies.

  16. Electrochemical DNA biosensor for detection of DNA damage induced by hydroxyl radicals.

    PubMed

    Hájková, Andrea; Barek, Jiří; Vyskočil, Vlastimil

    2017-08-01

    A simple electrochemical DNA biosensor based on a glassy carbon electrode (GCE) was prepared by adsorbing double-stranded DNA (dsDNA) onto the GCE surface and subsequently used for the detection of dsDNA damage induced by hydroxyl radicals. Investigation of the mutual interaction between hydroxyl radicals and dsDNA was conducted using a combination of several electrochemical detection techniques: square-wave voltammetry for direct monitoring the oxidation of dsDNA bases, and cyclic voltammetry and electrochemical impedance spectroscopy as indirect electrochemical methods making use of the redox-active indicator [Fe(CN)6](4-/3-). Hydroxyl radicals were generated electrochemically on the surface of a boron-doped diamond electrode and chemically (via the Fenton's reaction or the auto-oxidation of Fe(II)). The extent of dsDNA damage by electrochemically generated hydroxyl radicals depended on the current density applied to the generating electrode: by applying 5, 10, and 50mAcm(-2), selected relative biosensor responses decreased after 3min incubation from 100% to 38%, 27%, and 3%, respectively. Chemically generated hydroxyl radicals caused less pronounced dsDNA damage, and their damaging activity depended on the form of Fe(II) ions: decreases to 49% (Fenton's reaction; Fe(II) complexed with EDTA) and 33% (auto-oxidation of Fe(II); Fe(II) complexed with dsDNA) were observed after 10min incubation. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Chemistry and Structural Biology of DNA Damage and Biological Consequences

    PubMed Central

    Stone, Michael P.; Huang, Hai; Brown, Kyle L.; Shanmugam, Ganesh

    2013-01-01

    The formation of adducts by the reaction of chemicals with DNA is a critical step for the initiation of carcinogenesis. The structural analysis of various DNA adducts reveals that conformational and chemical rearrangements and interconversions are a common theme. Conformational changes are modulated both by the nature of adduct and the base sequences neighboring the lesion sites. Equilibria between conformational states may modulate both DNA repair and error-prone replication past these adducts. Likewise, chemical rearrangements of initially formed DNA adducts are also modulated both by the nature of adducts and the base sequences neighboring the lesion sites. In this review, we focus on DNA damage caused by a number of environmental and endogenous agents, and biological consequences. PMID:21922653

  18. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    2015-01-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  19. DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-Replication

    DTIC Science & Technology

    2005-04-01

    ml a that sustained rereplication leads to a dramatic decrease factor. Samples were fixed in 67% ethanol (vol/vol), washed twice with PBS, and...significant decrease in cell viability and a cellular DNA damage response. Strikingly, we have observed DNA damage in the absence of a classical...genome re-replicates. In this reporting period, we have shown that re-replication induces a rapid and significant decrease in cell viability and a

  20. How do male germ cells handle DNA damage?

    SciTech Connect

    Olsen, Ann-Karin; Lindeman, Birgitte; Wiger, Richard; Duale, Nur; Brunborg, Gunnar . E-mail: gunnar.brunborg@fhi.no

    2005-09-01

    Male reproductive health has received considerable attention in recent years. In addition to declining sperm quality, fertility problems and increased incidence of testicular cancer, there is accumulating evidence that genetic damage, in the form of unrepaired DNA lesions or de novo mutations, may be transmitted via sperm to the offspring. Such genetic damage may arise from environmental exposure or via endogenously formed reactive species, in stem cells or during spermatogenesis. Damaged testicular cells not removed by apoptosis rely on DNA repair for their genomic integrity to be preserved. To identify factors with potentially harmful effects on testicular cells and to characterise associated risk, a thorough understanding of repair mechanisms in these cells is of particular importance. Based on results from our own and other laboratories, we discuss the current knowledge of different pathways of excision repair in rodent and human testicular cells. It has become evident that, in human spermatogenic cells, some repair functions are indeed non-functional.

  1. Phototoxicity mechanisms: chlorpromazine photosensitized damage to DNA and cell membranes

    SciTech Connect

    Kochevar, K.E.

    1981-07-01

    Photosensitized damage to biological molecules is the initial process in phototoxic responses. It is now recognized that many phototoxic compounds can photosensitize damage to more than one type of biological substrate. The in vitro light-initiated reactions of phototoxic compounds with DNA, soluble proteins and membrane components can be classified by their molecular mechanisms: (1) those in which an excited state of the phototoxic compound (or an unstable species derived from it) reacts directly with the biological substrate and (2) those in which a molecule derived from the phototoxic compound (a photoproduct or an activated oxygen species) reacts with the biological substrate. This paper describes the mechanisms by which chlorpromazine photosensitizes damage to membranes, protein and DNA and compares them to the mechanisms of photosensitization by psoralens, porphyrins, dyes, and other molecules.

  2. Bile-Induced DNA Damage in Salmonella enterica

    PubMed Central

    Prieto, Ana I.; Ramos-Morales, Francisco; Casadesús, Josep

    2004-01-01

    In the absence of DNA adenine methylase, growth of Salmonella enterica serovar Typhimurium is inhibited by bile. Mutations in any of the mutH, mutL, and mutS genes suppress bile sensitivity in a Dam− background, indicating that an active MutHLS system renders Dam− mutants bile sensitive. However, inactivation of the MutHLS system does not cause bile sensitivity. An analogy with Escherichia coli, in which the MutHLS system sensitizes Dam− mutants to DNA-injuring agents, suggested that bile might cause DNA damage. In support of this hypothesis, we show that bile induces the SOS response in S. enterica and increases the frequency of point mutations and chromosomal rearrangements. Mutations in mutH, mutL, or mutS cause partial relief of virulence attenuation in a Dam− background (50- to 100-fold by the oral route and 10-fold intraperitoneally), suggesting that an active MutHLS system reduces the ability of Salmonella Dam− mutants to cope with DNA-damaging agents (bile and others) encountered during the infection process. The DNA-damaging ability of bile under laboratory conditions raises the possibility that the phenomenon may be relevant in vivo, since high bile concentrations are found in the gallbladder, the niche for chronic Salmonella infections. PMID:15611156

  3. Histone deacetylase regulation of ATM-mediated DNA damage signaling.

    PubMed

    Thurn, K Ted; Thomas, Scott; Raha, Paromita; Qureshi, Ian; Munster, Pamela N

    2013-10-01

    Ataxia-telangiectasia mutated (ATM) is a major regulator of the DNA damage response. ATM promotes the activation of BRCA1, CHK2, and p53 leading to the induction of response genes such as CDKN1A (p21), GADD45A, and RRM2B that promote cell-cycle arrest and DNA repair. The upregulation of these response genes may contribute to resistance of cancer cells to genotoxic therapies. Here, we show that histone deacetylases (HDAC) play a major role in mitigating the response of the ATM pathway to DNA damage. HDAC inhibition decreased ATM activation and expression, and attenuated the activation of p53 in vitro and in vivo. Select depletion of HDAC1 and HDAC2 was sufficient to modulate ATM activation, reduce GADD45A and RRM2B induction, and increase sensitivity to DNA strand breaks. The regulation of ATM by HDAC enzymes therefore suggests a vital role for HDAC1 and HDAC2 in the DNA damage response, and the potential use of the ATM pathway as a pharmacodynamic marker for combination therapies involving HDAC inhibitors. ©2013 AACR.

  4. Clustered DNA damage on subcellular level: effect of scavengers.

    PubMed

    Pachnerová Brabcová, Kateřina; Sihver, Lembit; Yasuda, Nakahiro; Matuo, Youichirou; Stěpán, Václav; Davídková, Marie

    2014-11-01

    Clustered DNA damages are induced by ionizing radiation, particularly of high linear energy transfer (LET). Compared to isolated DNA damage sites, their biological effects can be more severe. We investigated a clustered DNA damage induced by high LET radiation (C 290 MeV u(-1) and Fe 500 MeV u(-1)) in pBR322 plasmid DNA. The plasmid is dissolved in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers.

  5. Diseases Associated with Defective Responses to DNA Damage

    PubMed Central

    O’Driscoll, Mark

    2012-01-01

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

  6. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  7. Sunlight exposure-mediated DNA damage in young adults.

    PubMed

    Kato, Masashi; Iida, Machiko; Goto, Yuji; Kondo, Takaaki; Yajima, Ichiro

    2011-08-01

    Previous experimental studies showed that single ultraviolet B (UVB) light irradiation increased levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a well-established biomarker of carcinogenesis and oxidative DNA damage, in epithelial cells in animals and humans. We conducted for the first time an epidemiologic study to investigate the correlations among levels of oxidative DNA damage, skin pigmentation, and sunlight exposure in human daily life. Digitalized skin pigmentation levels and creatinine-adjusted urinary 8-OHdG levels were examined in 127 healthy young adults aged 20 to 24 years and in hairless mice with normal pigmented skin (HL-mice; n = 20) and hyperpigmented skin (HL-HPS-mice; n = 20). Data obtained by a questionnaire were also analyzed for the 127 subjects. Binary logistic regression analysis showed that increased sunlight intensity, but not sunlight-exposed time or sunlight-exposed skin area, was correlated with elevation in creatinine-adjusted urinary 8-OHdG levels. In contrast, increased skin pigmentation level, but not the use of sunscreen, was correlated with reduction in urinary 8-OHdG level in humans. UVB irradiation corresponding to several minutes of sunlight exposure significantly increased urinary 8-OHdG levels in HL-mice but not in HL-HPS-mice. We showed that increase in intensity of sunlight in human daily life increased levels of DNA damage. We also showed a protective effect of skin pigmentation on sunlight exposure-mediated DNA damage. We have provided more reliable evidence of routine sunlight exposure-mediated DNA damage in humans through the combination of epidemiologic and experimental studies. ©2011 AACR.

  8. DNA Damage: A Main Determinant of Vascular Aging

    PubMed Central

    Bautista-Niño, Paula K.; Portilla-Fernandez, Eliana; Vaughan, Douglas E.; Danser, A. H. Jan; Roks, Anton J. M.

    2016-01-01

    Vascular aging plays a central role in health problems and mortality in older people. Apart from the impact of several classical cardiovascular risk factors on the vasculature, chronological aging remains the single most important determinant of cardiovascular problems. The causative mechanisms by which chronological aging mediates its impact, independently from classical risk factors, remain to be elucidated. In recent years evidence has accumulated that unrepaired DNA damage may play an important role. Observations in animal models and in humans indicate that under conditions during which DNA damage accumulates in an accelerated rate, functional decline of the vasculature takes place in a similar but more rapid or more exaggerated way than occurs in the absence of such conditions. Also epidemiological studies suggest a relationship between DNA maintenance and age-related cardiovascular disease. Accordingly, mouse models of defective DNA repair are means to study the mechanisms involved in biological aging of the vasculature. We here review the evidence of the role of DNA damage in vascular aging, and present mechanisms by which genomic instability interferes with regulation of the vascular tone. In addition, we present potential remedies against vascular aging induced by genomic instability. Central to this review is the role of diverse types of DNA damage (telomeric, non-telomeric and mitochondrial), of cellular changes (apoptosis, senescence, autophagy), mediators of senescence and cell growth (plasminogen activator inhibitor-1 (PAI-1), cyclin-dependent kinase inhibitors, senescence-associated secretory phenotype (SASP)/senescence-messaging secretome (SMS), insulin and insulin-like growth factor 1 (IGF-1) signaling), the adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-nuclear factor kappa B (NFκB) axis, reactive oxygen species (ROS) vs. endothelial nitric oxide synthase (eNOS)-cyclic guanosine monophosphate (c

  9. DNA Damage: A Main Determinant of Vascular Aging.

    PubMed

    Bautista-Niño, Paula K; Portilla-Fernandez, Eliana; Vaughan, Douglas E; Danser, A H Jan; Roks, Anton J M

    2016-05-18

    Vascular aging plays a central role in health problems and mortality in older people. Apart from the impact of several classical cardiovascular risk factors on the vasculature, chronological aging remains the single most important determinant of cardiovascular problems. The causative mechanisms by which chronological aging mediates its impact, independently from classical risk factors, remain to be elucidated. In recent years evidence has accumulated that unrepaired DNA damage may play an important role. Observations in animal models and in humans indicate that under conditions during which DNA damage accumulates in an accelerated rate, functional decline of the vasculature takes place in a similar but more rapid or more exaggerated way than occurs in the absence of such conditions. Also epidemiological studies suggest a relationship between DNA maintenance and age-related cardiovascular disease. Accordingly, mouse models of defective DNA repair are means to study the mechanisms involved in biological aging of the vasculature. We here review the evidence of the role of DNA damage in vascular aging, and present mechanisms by which genomic instability interferes with regulation of the vascular tone. In addition, we present potential remedies against vascular aging induced by genomic instability. Central to this review is the role of diverse types of DNA damage (telomeric, non-telomeric and mitochondrial), of cellular changes (apoptosis, senescence, autophagy), mediators of senescence and cell growth (plasminogen activator inhibitor-1 (PAI-1), cyclin-dependent kinase inhibitors, senescence-associated secretory phenotype (SASP)/senescence-messaging secretome (SMS), insulin and insulin-like growth factor 1 (IGF-1) signaling), the adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-nuclear factor kappa B (NFκB) axis, reactive oxygen species (ROS) vs. endothelial nitric oxide synthase (eNOS)-cyclic guanosine monophosphate (c

  10. Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response.

    PubMed

    Quanz, Maria; Chassoux, Danielle; Berthault, Nathalie; Agrario, Céline; Sun, Jian-Sheng; Dutreix, Marie

    2009-07-21

    Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an "all-or-none" pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a "false" DNA damage signaling that disorganizes the DNA repair system.

  11. The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.

    PubMed

    Kemp, Michael G; Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

  12. Microfluidic array for simultaneous detection of DNA oxidation and DNA-adduct damage.

    PubMed

    Song, Boya; Shen, Min; Jiang, Di; Malla, Spundana; Mosa, Islam M; Choudhary, Dharamainder; Rusling, James F

    2016-10-21

    Exposure to chemical pollutants and pharmaceuticals may cause health issues caused by metabolite-related toxicity. This paper reports a new microfluidic electrochemical sensor array with the ability to simultaneously detect common types of DNA damage including oxidation and nucleobase adduct formation. Sensors in the 8-electrode screen-printed carbon array were coated with thin films of metallopolymers osmium or ruthenium bipyridyl-poly(vinylpyridine) chloride (OsPVP, RuPVP) along with DNA and metabolic enzymes by layer-by-layer electrostatic assembly. After a reaction step in which test chemicals and other necessary reagents flow over the array, OsPVP selectively detects oxidized guanines on the DNA strands, and RuPVP detects DNA adduction by metabolites on nucleobases. We demonstrate array performance for test chemicals including 17β-estradiol (E2), its metabolites 4-hydroxyestradiol (4-OHE2), 2-hydroxyestradiol (2-OHE2), catechol, 2-nitrosotoluene (2-NO-T), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 2-acetylaminofluorene (2-AAF). Results revealed DNA-adduct and oxidation damage in a single run to provide a metabolic-genotoxic chemistry screen. The array measures damage directly in unhydrolyzed DNA, and is less expensive, faster, and simpler than conventional methods to detect DNA damage. The detection limit for oxidation is 672 8-oxodG per 10(6) bases. Each sensor requires only 22 ng of DNA, so the mass detection limit is 15 pg (∼10 pmol) 8-oxodG.

  13. Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells.

    PubMed

    Leadon, S A; Sumerel, J; Minton, T A; Tischler, A

    1995-12-01

    In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.

  14. DNA precipitation assay: a rapid and simple method for detecting DNA damage in mammalian cells

    SciTech Connect

    Olive, P.L.

    1988-01-01

    When mammalian cells are lysed in 2% sodium dodecyl sulphate detergent followed by addition of an equal volume of 0.12 M potassium chloride, a precipitate forms that can be collected by low-speed centrifugation. This precipitate contains the cell protein and nucleic acid in close association with protein. In the absence of DNA damage, most of the DNA precipitates, but when DNA strand breaks are created by exposing cells to ionizing radiation or toxic chemicals, DNA is released from the protein and remains in the supernatant after centrifugation. The proportion of DNA remaining in the supernatant is thus a measure of the amount of DNA damage. This assay is characterized in terms of optimum cell number and pH and dose-response curves for DNA damage and cell survival following ionizing radiation, MNNG, BCNU, and VP-16 are shown. Sensitivity, simplicity, speed, and large sample handling capacity should allow wide application of this new assay to a variety of questions concerning DNA damage and repair.

  15. Measurement of oxidatively generated base damage in cellular DNA.

    PubMed

    Cadet, Jean; Douki, Thierry; Ravanat, Jean-Luc

    2011-06-03

    This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.

  16. Taking a Bad Turn: Compromised DNA Damage Response in Leukemia

    PubMed Central

    Nilles, Nadine; Fahrenkrog, Birthe

    2017-01-01

    Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB) and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK) and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2), key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS), and isocitrate dehydrogenase 1 and 2 (IDH1/2). A detailed understanding of the basis for defective DNA damage response (DDR) mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients. PMID:28471392

  17. Carbon dots based FRET for the detection of DNA damage.

    PubMed

    Kudr, Jiri; Richtera, Lukas; Xhaxhiu, Kledi; Hynek, David; Heger, Zbynek; Zitka, Ondrej; Adam, Vojtech

    2017-02-09

    Here, we aimed our attention at the synthesis of carbon dots (C-dots) with the ability to interact with DNA to suggest an approach for the detection of DNA damage. Primarily, C-dots modified with amine moieties were synthesized using the one-step microwave pyrolysis of citric acid in the presence of diethylenetriamine. The C-dots showed strong photoluminescence with a quantum yield of 4%. In addition, the C-dots (2.8±0.8nm) possessed a good colloidal stability and exhibited a positive surface charge (ζ=36mV) at a neutral pH. An interaction study of the C-dots and the DNA fragment of λ bacteriophage was performed, and the DNA binding resulted in changes to the photoluminescent and absorption properties of the C-dots. A binding of the C-dots to DNA was also observed as a change to DNA electrophoretic mobility and a decreased ability to intercalate ethidium bromide (EtBr). Moreover, the Förster (or fluorescence) resonance energy transfer (FRET) between the C-dots and EtBr was studied, in which the C-dots serve as an excitation energy donor and the EtBr serves as an acceptor. When DNA was damaged using ultraviolet (UV) radiation (λ=254nm) and hydroxyl radicals, the intensity of the emitted photoluminescence at 612nm significantly decreased. The concept was proved on analysis of the genomic DNA from PC-3 cells and DNA isolated from melanoma tissues.

  18. DNA damage response induced by HZE particles in human cells

    NASA Astrophysics Data System (ADS)

    Chen, David; Aroumougame, Asaithamby

    Convincing evidences indicate that high-linear energy transfer (LET) ionizing radiation (IR) induced complex DNA lesions are more difficult to repair than isolated DNA lesions induced by low-LET IR; this has been associated with the increased RBE for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in high energy charged-particle irradiated human cells. We have employed an in situ method to directly monitor induction and repair of clustered DNA lesions at the single-cell level. 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 determined the cellular ability to repair these damages. Importantly, examination of metaphase cells derived from HZE particle irradiated cells revealed that the extent of chromosome aberrations directly correlated with the levels of unrepaired clustered DNA lesions. In addition, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We found that complex DNA lesions induced by HZE particles were even more difficult to be repaired in organotypic 3D culture, resulting enhanced cell killing and chromosome aberrations. Our data suggest that DNA repair capability in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis. As the organotypic 3D model mimics human lung, it opens up new experimental approaches to explore the effect of radiation in vivo and will have important implications for evaluating radiation risk in human tissues.

  19. Situation-dependent repair of DNA damage in yeast

    SciTech Connect

    von Borstel, R.C.; Hastings, P.J.

    1985-01-01

    The concept of channelling of lesions in DNA into defined repair systems has been used to explain many aspects of induced and spontaneous mutation. The channelling hypothesis states that lesions excluded from one repair process will be taken up by another repair process. This is a simplification. The three known modes of repair of damage induced by radiation are not equivalent modes of repair; they are, instead, different solutions to the problem of replacement of damaged molecules with new molecules which have the same informational content as those that were damaged. The mode of repair that is used is the result of the response to the situation in which the damage takes place. Thus, when the most likely mode of repair does not take place, then the situation changes with respect to the repair of the lesion; the lesion may enter the replication fork and be reparable by another route.

  20. Bleomycin-induced alterations in DNA replication: relationship to DNA damage.

    PubMed

    Dziegielewski, J; Melendy, T; Beerman, T A

    2001-01-23

    Bleomycin (BLM), a well-known DNA scission agent, is assumed to inhibit intracellular DNA replication by damaging the DNA template (cis-acting mechanism), although other DNA damaging compounds can alter DNA replication through modulation of crucial replication factor(s) (trans-acting mechanism). The present study examines the relationship between DNA damage and inhibition of replication caused by BLM in the well-defined simian virus 40 (SV40) intracellular and cell-free in vitro systems. Treatment of SV40-infected BSC-1 cells for 2 h with BLM at 50 microg/mL, induced 0.3 break/viral genome. Under the same treatment conditions, analysis of replication intermediates on two-dimensional gels showed a decrease in both mass of SV40 replication intermediates and replication activity. The mass of SV40 intermediates was decreased to about 30%, whereas replication activity was reduced to less than 5%. These results suggest that BLM inhibits both initiation and elongation phases of SV40 replication. In a cell-free DNA replication system, extracts from BLM-treated cells (50 micro/mL) were able to support SV40 DNA replication by only 50%. In this study, non-drug-treated DNA template was used, implying that BLM can induce a trans-acting effect. Finally, the drug-induced effects on SV40 DNA replication in cell-free and intracellular viral systems were compared to the effects on genomic DNA replication in BSC-1 cells. Overall, the results support the concept that BLM-induced inhibition of DNA replication occurs by both trans- (inhibition of replication of nondamaged template) and cis-acting mechanisms (template damage).

  1. Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

    PubMed Central

    Mallik, Sarita; Popodi, Ellen M.; Hanson, Andrew J.

    2015-01-01

    ABSTRACT Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. IMPORTANCE DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings

  2. DNA repair and the evolution of transformation in Bacillus subtilis. 3. Sex with damaged DNA

    SciTech Connect

    Hoelzer, M.A.; Michod, R.E. )

    1991-06-01

    Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. The authors document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome.

  3. Targeting ATR in DNA damage response and cancer therapeutics.

    PubMed

    Fokas, Emmanouil; Prevo, Remko; Hammond, Ester M; Brunner, Thomas B; McKenna, W Gillies; Muschel, Ruth J

    2014-02-01

    The ataxia telangiectasia and Rad3-related (ATR) plays an important role in maintaining genome integrity during DNA replication through the phosphorylation and activation of Chk1 and regulation of the DNA damage response. Preclinical studies have shown that disruption of ATR pathway can exacerbate the levels of replication stress in oncogene-driven murine tumors to promote cell killing. Additionally, inhibition of ATR can sensitise tumor cells to radiation or chemotherapy. Accumulating evidence suggests that targeting ATR can selectively sensitize cancer cells but not normal cells to DNA damage. Furthermore, in hypoxic conditions, ATR blockade results in overloading replication stress and DNA damage response causing cell death. Despite the attractiveness of ATR inhibition in the treatment of cancer, specific ATR inhibitors have remained elusive. In the last two years however, selective ATR inhibitors suitable for in vitro and - most recently - in vivo studies have been identified. In this article, we will review the literature on ATR function, its role in DDR and the potential of ATR inhibition to enhance the efficacy of radiation and chemotherapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Intracellular DNA damage produced by a series of diacridines.

    PubMed Central

    Roos, I A; Wakelin, L P; Henry, S J

    1985-01-01

    The intracellular DNA damage produced by a series of diacridines after a 2 h pulse treatment of L1210 cells in culture was investigated by using the alkaline-elution technique. Like other intercalating agents, diacridines produce single-strand breaks and protein-DNA links. There is a large increase in both types of damage as the alkane chain linking the two 9-aminoacridine residues is increased beyond five methylene groups, which is consistent with the previously observed change from monofunctional to bifunctional intercalation [Wakelin, Romanos, Chen, Glaubiger, Canellakis & Waring (1978) Biochemistry 17, 5057-5063]. For linker chains of less than six methylene groups these agents produce less DNA damage than does the parent 9-aminoacridine at the same drug concentration. Unlike the monofunctional intercalators previously investigated [Ross, Glaubiger & Kohn (1979) Biochim. Biophys. Acta 562, 41-50; Zwelling, Michaels, Erickson, Ungerleider, Nichols & Kohn (1981) Biochemistry 20, 6553-6563; Zwelling, Kerrigan & Michaels (1982) Cancer Res. 42, 2687-2691; Zwelling, Michaels, Kerrigan, Pommier & Kohn (1982) Biochem. Pharmacol. 31, 3261-3267], there is no correlation between the number of single-strand breaks and protein-DNA links produced by these diacridines. PMID:3977863

  5. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    PubMed

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-01-18

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer.

  6. Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

    NASA Astrophysics Data System (ADS)

    Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

    2014-01-01

    DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

  7. Reduction in oxidatively generated DNA damage following smoking cessation

    PubMed Central

    2011-01-01

    Background Cigarette smoking is a known cause of cancer, and cancer may be in part due to effects of oxidative stress. However, whether smoking cessation reverses oxidatively induced DNA damage unclear. The current study sought to examine the extent to which three DNA lesions showed significant reductions after participants quit smoking. Methods Participants (n = 19) in this study were recruited from an ongoing 16-week smoking cessation clinical trial and provided blood samples from which leukocyte DNA was extracted and assessed for 3 DNA lesions (thymine glycol modification [d(TgpA)]; formamide breakdown of pyrimidine bases [d(TgpA)]; 8-oxo-7,8-dihydroguanine [d(Gh)]) via liquid chromatography tandem mass spectrometry (LC-MS/MS). Change in lesions over time was assessed using generalized estimating equations, controlling for gender, age, and treatment condition. Results Overall time effects for the d(TgpA) (χ2(3) = 8.068, p < 0.045), d(PfpA) (χ2(3) = 8.477, p < 0.037), and d(Gh) (χ2(3) = 37.599, p < 0.001) lesions were seen, indicating levels of each decreased significantly after CO-confirmed smoking cessation. The d(TgpA) and d(PfpA) lesions show relatively greater rebound at Week 16 compared to the d(Gh) lesion (88% of baseline for d(TgpA), 64% of baseline for d(PfpA), vs 46% of baseline for d(Gh)). Conclusions Overall, results from this analysis suggest that cigarette smoking contributes to oxidatively induced DNA damage, and that smoking cessation appears to reduce levels of specific damage markers between 30-50 percent in the short term. Future research may shed light on the broader array of oxidative damage influenced by smoking and over longer durations of abstinence, to provide further insights into mechanisms underlying carcinogenesis. PMID:21569419

  8. Skeletal Muscle DNA Damage Precedes Spinal Motor Neuron DNA Damage in a Mouse Model of Spinal Muscular Atrophy (SMA)

    PubMed Central

    Fayzullina, Saniya; Martin, Lee J.

    2014-01-01

    Spinal Muscular Atrophy (SMA) is a hereditary childhood disease that causes paralysis by progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. The mechanisms by which lack of SMN causes SMA pathology are not known, making it very difficult to develop effective therapies. We investigated whether DNA damage is a perinatal pathological event in SMA, and whether DNA damage and cell death first occur in skeletal muscle or spinal cord of SMA mice. We used a mouse model of severe SMA to ascertain the extent of cell death and DNA damage throughout the body of prenatal and newborn mice. SMA mice at birth (postnatal day 0) exhibited internucleosomal fragmentation in genomic DNA from hindlimb skeletal muscle, but not in genomic DNA from spinal cord. SMA mice at postnatal day 5, compared with littermate controls, exhibited increased apoptotic cell death profiles in skeletal muscle, by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling, and electron microscopy. SMA mice had no increased cell death, no loss of choline acetyl transferase (ChAT)-positive motor neurons, and no overt pathology in the ventral horn of the spinal cord. At embryonic days 13 and 15.5, SMA mice did not exhibit statistically significant increases in cell death profiles in spinal cord or skeletal muscle. Motor neuron numbers in the ventral horn, as identified by ChAT immunoreactivity, were comparable in SMA mice and control littermates at embryonic day 15.5 and postnatal day 5. These observations demonstrate that in SMA, disease in skeletal muscle emerges before pathology in spinal cord, including loss of motor neurons. Overall, this work identifies DNA damage and cell death in skeletal muscle as therapeutic targets for SMA. PMID:24667816

  9. Skeletal muscle DNA damage precedes spinal motor neuron DNA damage in a mouse model of Spinal Muscular Atrophy (SMA).

    PubMed

    Fayzullina, Saniya; Martin, Lee J

    2014-01-01

    Spinal Muscular Atrophy (SMA) is a hereditary childhood disease that causes paralysis by progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. The mechanisms by which lack of SMN causes SMA pathology are not known, making it very difficult to develop effective therapies. We investigated whether DNA damage is a perinatal pathological event in SMA, and whether DNA damage and cell death first occur in skeletal muscle or spinal cord of SMA mice. We used a mouse model of severe SMA to ascertain the extent of cell death and DNA damage throughout the body of prenatal and newborn mice. SMA mice at birth (postnatal day 0) exhibited internucleosomal fragmentation in genomic DNA from hindlimb skeletal muscle, but not in genomic DNA from spinal cord. SMA mice at postnatal day 5, compared with littermate controls, exhibited increased apoptotic cell death profiles in skeletal muscle, by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling, and electron microscopy. SMA mice had no increased cell death, no loss of choline acetyl transferase (ChAT)-positive motor neurons, and no overt pathology in the ventral horn of the spinal cord. At embryonic days 13 and 15.5, SMA mice did not exhibit statistically significant increases in cell death profiles in spinal cord or skeletal muscle. Motor neuron numbers in the ventral horn, as identified by ChAT immunoreactivity, were comparable in SMA mice and control littermates at embryonic day 15.5 and postnatal day 5. These observations demonstrate that in SMA, disease in skeletal muscle emerges before pathology in spinal cord, including loss of motor neurons. Overall, this work identifies DNA damage and cell death in skeletal muscle as therapeutic targets for SMA.

  10. p53 in the DNA damage repair process

    PubMed Central

    Williams, Ashley B.; Schumacher, Björn

    2016-01-01

    The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA damage response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA repair systems. It thus appears as if p53 is multitasking in protecting from cancer development by maintaining genome stability. PMID:27048304

  11. DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa)

    SciTech Connect

    Ammermann, D.

    1988-05-01

    Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of (6-/sup 3/H)thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.

  12. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    PubMed Central

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells. PMID:26666690

  13. DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage.

    PubMed

    Suji, G; Sivakami, S

    2007-11-01

    Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.

  14. Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900MHz radiofrequency fields.

    PubMed

    Sun, Yulong; Zong, Lin; Gao, Zhen; Zhu, Shunxing; Tong, Jian; Cao, Yi

    2017-03-01

    HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900MHz radiofrequency fields (RF) at 120μW/cm(2) power intensity for 4h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2'-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Homologous recombination is required for recovery from oxidative DNA damage.

    PubMed

    Hayashi, Michio; Umezu, Keiko

    2017-04-03

    We have been studying the genetic events, including chromosome loss, chromosome rearrangements and intragenic point mutations, that are responsible for the deletion of a URA3 marker in a loss of heterozygosity (LOH) assay in the yeast Saccharomycess cerevisiae. With this assay, we previously showed that homologous recombination plays an important role in genome maintenance in response to DNA lesions that occur spontaneously in normally growing cells. Here, to investigate DNA lesions capable of triggering homologous recombination, we examined the effects of oxidative stress, a prominent cause of endogenous DNA damage, on LOH events. Treatment of log-phase cells with H2O2 first caused growth arrest and then, during the subsequent recovery, chromosome loss and various chromosome rearrangements were induced more than 10-fold. Further analysis of the rearrangements showed that gene conversion was strongly induced, approximately 100 times more frequently than in untreated cells. Consistent with these results, two diploid strains deficient for homologous recombination, rad52Δ/rad52Δ and rad51Δ/rad51Δ, were sensitive to H2O2 treatment. In addition, chromosome DNA breaks were detected in H2O2-treated cells using pulsed-field gel electrophoresis. Altogether, these results suggest that oxidative stress induced recombinogenic lesions on chromosomes, which then triggered homologous recombination leading to chromosome rearrangements, and that this response contributed to the survival of cells afflicted by oxidative DNA damage. We therefore conclude that homologous recombination is required for the recovery of cells from oxidative stress.

  16. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    SciTech Connect

    Huselton, C.A.; Hill, H.Z. )

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals.

  17. Gilvocarcin V, a Photodynamic DNA Damaging Agent Of Unusual Potency

    NASA Astrophysics Data System (ADS)

    Elespuru, Rosalie K.; Look, Sally A.

    1988-02-01

    Gilvocarcin V (GV) is a planar, aromatic DNA-intercalating C-glycoside isolated as a natural product antibiotic. In the presence of UVA or visible radiation, it becomes a DNA damaging agent at low doses in both bacterial and mammalian cells. In mice treated without regard to light exposure, GV exhibited antitumor activity at high doses, with little accompanying toxicity. Wavelength-dependence studies showed that lambda prophage induction profiles were similar to (part of) the absorption spectrum of GV, with a peak near 400 nm. However, significant induction at a higher wavelength (546 nm), was observed at relatively high (e.g. 1 μg/m1) concentrations of GV. The DNA damaging activity of GV was dependent on both the concentration of antibiotic and the fluence of radiation in a reciprocal manner. Mutagenesis and DNA binding experiments suggest a preference for interaction with AT-rich regions of DNA, but multiple modes of interaction seem likely. The presence of different C-glycosides on the gilvocarcin V chromophore may alter the pharmacological properties of the molecule, but photoactivation appears to be independent of these groups. The therapeutic possibilities of gilvocarcins remain largely unexplored; the demonstrated potency of these compounds when activated, the reciprocity effect, possibility of structural variation, and apparent lack of toxicity in mammalian systems are properties which could be exploited in therapeutic development.

  18. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells.

    PubMed

    Huselton, C A; Hill, H Z

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals.

  19. Primary DNA damage in chrome-plating workers.

    PubMed

    Gambelunghe, A; Piccinini, R; Ambrogi, M; Villarini, M; Moretti, M; Marchetti, C; Abbritti, G; Muzi, G

    2003-06-30

    In order to evaluate the primary DNA damage due to occupational exposure to chromium (VI), DNA strand-breaks and apoptosis in peripheral lymphocytes were measured in a group of 19 chrome-plating workers. DNA strand-breaks was assessed by alkaline (pH>13) single-cell microgel electrophoresis ('comet') assay, while apoptosis was measured by flow-cytometry after propidium iodide staining of the cells. Concentrations of chromium in urine, erythrocytes and lymphocytes were investigated as biological indicators of exposure. A group of 18 hospital workers (control group I) and another 20 university personnel (control group II) without exposure to chromium were also studied as controls. The results of the study show that chrome-plating workers have higher levels of chromium in urine, erythrocytes and lymphocytes than unexposed workers. Comet tail moment values, assumed as index of DNA damage, are increased in chromium-exposed workers and results are significantly correlated to chromium lymphocyte concentrations. No difference emerged in the percentage of apoptotic nuclei in exposed and unexposed workers. The study confirms that measurements of chromium in erythrocytes and lymphocytes may provide useful information about recent and past exposure to hexavalent chromium at the workplace. The increase in DNA strand-breaks measured by comet assay suggests this test is valid for the biological monitoring of workers exposed to genotoxic compounds such as chromium (VI).

  20. DNA damage in mammalian cells following heavy-ion irradiation

    SciTech Connect

    Rosander, K.; Frankel, K.A.; Cerda, H.; Phillips, M.H.; Lo, E.H.; Fabrikant, I.; Fabrikant, J.I.; Levy, R.P.

    1989-09-01

    In our laboratory we have been investigating DNA damage and repair in the endothelial and oligodendroglial cells of the mouse brain after irradiation using two different types of heavy ions, helium and neon. The method used, the unwinding technique with subsequent staining of the DNA with acridine orange, has been proven to be useful for nondividing cells and analysis using a microscope photometric technique. Our primary goal has been to obtain a measure of RBE, in the dose range used in clinical treatment of various brain disorders using heavy charged particle radiosurgery. 12 refs., 5 figs.

  1. Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions.

    PubMed

    Tobias, Frank; Löb, Daniel; Lengert, Nicor; Durante, Marco; Drossel, Barbara; Taucher-Scholz, Gisela; Jakob, Burkhard

    2013-01-01

    The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the

  2. Preservation of ancient DNA in thermally damaged archaeological bone

    NASA Astrophysics Data System (ADS)

    Ottoni, Claudio; Koon, Hannah E. C.; Collins, Matthew J.; Penkman, Kirsty E. H.; Rickards, Olga; Craig, Oliver E.

    2009-02-01

    Evolutionary biologists are increasingly relying on ancient DNA from archaeological animal bones to study processes such as domestication and population dispersals. As many animal bones found on archaeological sites are likely to have been cooked, the potential for DNA preservation must be carefully considered to maximise the chance of amplification success. Here, we assess the preservation of mitochondrial DNA in a medieval cattle bone assemblage from Coppergate, York, UK. These bones have variable degrees of thermal alterations to bone collagen fibrils, indicative of cooking. Our results show that DNA preservation is not reliant on the presence of intact collagen fibrils. In fact, a greater number of template molecules could be extracted from bones with damaged collagen. We conclude that moderate heating of bone may enhance the retention of DNA fragments. Our results also indicate that ancient DNA preservation is highly variable, even within a relatively recent assemblage from contexts conducive to organic preservation, and that diagenetic parameters based on protein diagenesis are not always useful for predicting ancient DNA survival.

  3. Preservation of ancient DNA in thermally damaged archaeological bone.

    PubMed

    Ottoni, Claudio; Koon, Hannah E C; Collins, Matthew J; Penkman, Kirsty E H; Rickards, Olga; Craig, Oliver E

    2009-02-01

    Evolutionary biologists are increasingly relying on ancient DNA from archaeological animal bones to study processes such as domestication and population dispersals. As many animal bones found on archaeological sites are likely to have been cooked, the potential for DNA preservation must be carefully considered to maximise the chance of amplification success. Here, we assess the preservation of mitochondrial DNA in a medieval cattle bone assemblage from Coppergate, York, UK. These bones have variable degrees of thermal alterations to bone collagen fibrils, indicative of cooking. Our results show that DNA preservation is not reliant on the presence of intact collagen fibrils. In fact, a greater number of template molecules could be extracted from bones with damaged collagen. We conclude that moderate heating of bone may enhance the retention of DNA fragments. Our results also indicate that ancient DNA preservation is highly variable, even within a relatively recent assemblage from contexts conducive to organic preservation, and that diagenetic parameters based on protein diagenesis are not always useful for predicting ancient DNA survival.

  4. DNA damage profiles induced by sunlight at different latitudes.

    PubMed

    Schuch, André Passaglia; Yagura, Teiti; Makita, Kazuo; Yamamoto, Hiromasa; Schuch, Nelson Jorge; Agnez-Lima, Lucymara Fassarella; MacMahon, Ricardo Monreal; Menck, Carlos Frederico Martins

    2012-04-01

    Despite growing knowledge on the biological effects of ultraviolet (UV) radiation on human health and ecosystems, it is still difficult to predict the negative impacts of the increasing incidence of solar UV radiation in a scenario of global warming and climate changes. Hence, the development and application of DNA-based biological sensors to monitor the solar UV radiation under different environmental conditions is of increasing importance. With a mind to rendering a molecular view-point of the genotoxic impact of sunlight, field experiments were undertaken with a DNA-dosimeter system in parallel with physical photometry of solar UVB/UVA radiation, at various latitudes in South America. On applying biochemical and immunological approaches based on specific DNA-repair enzymes and antibodies, for evaluating sunlight-induced DNA damage profiles, it became clear that the genotoxic potential of sunlight does indeed vary according to latitude. Notwithstanding, while induction of oxidized DNA bases is directly dependent on an increase in latitude, the generation of 6-4PPs is inversely so, whereby the latter can be regarded as a biomolecular marker of UVB incidence. This molecular DNA lesion-pattern largely reflects the relative incidence of UVA and UVB energy at any specific latitude. Hereby is demonstrated the applicability of this DNA-based biosensor for additional, continuous field experiments, as a means of registering variations in the genotoxic impact of solar UV radiation. © 2012 Wiley Periodicals, Inc.

  5. Investigation of perfluorooctanoic acid induced DNA damage using electrogenerated chemiluminescence associated with charge transfer in DNA.

    PubMed

    Lu, Liping; Guo, Linqing; Li, Meng; Kang, Tianfang; Cheng, Shuiyuan; Miao, Wujian

    2016-10-01

    An electrogenerated chemiluminescence (ECL)-DNA sensor was designed and fabricated for the investigation of DNA damage by a potential environmental pollutant, perfluorooctanoic acid (PFOA). The ECL-DNA sensor consisted of a Au electrode that had a self-assembled monolayer of 15 base-pair double-stranded (ds) DNA oligonucleotides with covalently attached semiconductor CdSe quantum dots (QDs) at the distal end of the DNA. Characterization of the ECL-DNA sensor was conducted with X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), ECL, and cyclic voltammetry before and after the exposure of the sensor to PFOA. Consistent data revealed that the dsDNA on Au was severely damaged upon the incubation of the electrode in PFOA, causing significant increase in charge (or electron) transfer (CT) resistance within DNA strands. Consequently, the cathodic coreactant ECL responses of the Au/dsDNA-QDs electrode in the presence of K2S2O8 were markedly decreased. The strong interaction between DNA and PFOA via the hydrophobic interaction, especially the formation of F···H hydrogen bonds by insertion of the difluoro-methylene group of PFOA into the DNA base pairs, was believed to be responsible for the dissociation or loosening of dsDNA structure, which inhibited the CT through DNA. A linear relationship between the ECL signal of the sensor and the logarithmical concentration of PFOA displayed a dynamic range of 1.00 × 10(-14)-1.00 × 10(-4) M, with a limit of detection of 1.00 × 10(-15) M at a signal-to-noise ratio of 3. Graphical Abstract Illustration of ECL detection of PFOA on a Au/dsDNA-QDs ECL-DNA sensor.

  6. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  7. The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead.

    PubMed

    Lorenzo, Yolanda; Costa, Solange; Collins, Andrew R; Azqueta, Amaya

    2013-07-01

    DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity.

  8. DNA damage associated with mitosis and cytokinesis failure.

    PubMed

    Hayashi, M T; Karlseder, J

    2013-09-26

    Mitosis is a highly dynamic process, aimed at separating identical copies of genomic material into two daughter cells. A failure of the mitotic process generates cells that carry abnormal chromosome numbers. Such cells are predisposed to become tumorigenic upon continuous cell division and thus need to be removed from the population to avoid cancer formation. Cells that fail in mitotic progression indeed activate cell death or cell cycle arrest pathways; however, these mechanisms are not well understood. Growing evidence suggests that the formation of de novo DNA damage during and after mitotic failure is one of the causal factors that initiate those pathways. Here, we analyze several distinct malfunctions during mitosis and cytokinesis that lead to de novo DNA damage generation.

  9. Alternative splicing of DNA damage response genes and gastrointestinal cancers.

    PubMed

    Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio

    2014-12-14

    Alternative splicing, which is a common phenomenon in mammalian genomes, is a fundamental process of gene regulation and contributes to great protein diversity. Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer. In this review, we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis, focusing on the potential relationship of alternative splicing, DNA damage, and gastrointestinal cancers. We will also discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies.

  10. Rewiring of genetic networks in response to DNA damage.

    PubMed

    Bandyopadhyay, Sourav; Mehta, Monika; Kuo, Dwight; Sung, Min-Kyung; Chuang, Ryan; Jaehnig, Eric J; Bodenmiller, Bernd; Licon, Katherine; Copeland, Wilbert; Shales, Michael; Fiedler, Dorothea; Dutkowski, Janusz; Guénolé, Aude; van Attikum, Haico; Shokat, Kevan M; Kolodner, Richard D; Huh, Won-Ki; Aebersold, Ruedi; Keogh, Michael-Christopher; Krogan, Nevan J; Ideker, Trey

    2010-12-03

    Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli.

  11. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    PubMed

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide

    SciTech Connect

    Hagensee, M.E.; Bryan, S.K.; Moses, R.E.

    1987-10-01

    The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.

  13. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    SciTech Connect

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  14. Torin2 Suppresses Ionizing Radiation-Induced DNA Damage Repair.

    PubMed

    Udayakumar, Durga; Pandita, Raj K; Horikoshi, Nobuo; Liu, Yan; Liu, Qingsong; Wong, Kwok-Kin; Hunt, Clayton R; Gray, Nathanael S; Minna, John D; Pandita, Tej K; Westover, Kenneth D

    2016-05-01

    Several classes of inhibitors of the mammalian target of rapamycin (mTOR) have been developed based on its central role in sensing growth factor and nutrient levels to regulate cellular metabolism. However, its ATP-binding site closely resembles other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, resulting in reactivity with these targets that may also be therapeutically useful. The ATP-competitive mTOR inhibitor, Torin2, shows biochemical activity against the DNA repair-associated proteins ATM, ATR and DNA-PK, which raises the possibility that Torin2 and related compounds might radiosensitize cancerous tumors. In this study Torin2 was also found to enhance ionizing radiation-induced cell killing in conditions where ATM was dispensable, confirming the requirement for multiple PIKK targets. Moreover, Torin2 did not influence the initial appearance of γ-H2AX foci after irradiation but significantly delayed the disappearance of radiation-induced γ-H2AX foci, indicating a DNA repair defect. Torin2 increased the number of radiation-induced S-phase specific chromosome aberrations and reduced the frequency of radiation-induced CtIP and Rad51 foci formation, suggesting that Torin2 works by blocking homologous recombination (HR)-mediated DNA repair resulting in an S-phase specific DNA repair defect. Accordingly, Torin2 reduced HR-mediated repair of I-Sce1-induced DNA damage and contributed to replication fork stalling. We conclude that radiosensitization of tumor cells by Torin2 is associated with disrupting ATR- and ATM-dependent DNA damage responses. Our findings support the concept of developing combination cancer therapies that incorporate ionizing radiation therapy and Torin2 or compounds with similar properties.

  15. Pyrosequencing: Applicability for Studying DNA Damage-induced Mutagenesis

    PubMed Central

    Minko, Irina G.; Earley, Lauriel F.; Larlee, Kimberly E.; Lin, Ying-Chih; Lloyd, R. Stephen

    2014-01-01

    Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a predetermined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively-labeled probes, and verification of mutations by Sanger sequencing. In search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N6-(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5′-GCNGG-3′ sequence context. Pyrosequencing detected ~8% G to T transversions and ~3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure [Earley et al., 2013]. However, ~3.5% G to C transversions and ~2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ~1-2% for A and T and ~5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols. PMID:24962778

  16. Radiation Combined Injury: DNA Damage, Apoptosis, and Autophagy

    DTIC Science & Technology

    2010-01-01

    the course of their disease (5) represents another significant source of exposure as normal tissues are subjected to radiation injury. Those charged...that luminal microbiota com- position may influence the host’s intestinal response to radiation and may change in those developing postirradiation... disease . Annu. Rev. Pathol. Mecha. Dis. 3: 247-255, 2008. 41. Kurz, E.U. and Lees-Miller, S.P. DNA damage-induced activation of ATM and ATM

  17. Impaired DNA damage repair as a common feature of neurodegenerative diseases and psychiatric disorders.

    PubMed

    Shiwaku, H; Okazawa, H

    2015-01-01

    Impaired DNA damage repair is a common pathological endophenotype of some types of neurodegenerative diseases, intellectual disabilities, and psychiatric diseases. Dysfunctional DNA repair and DNA damage, including DNA double-stranded breaks, are linked to transcriptional dysfunction and abnormal DNA methylation. Impaired DNA repair in neural stem cells leads to microcephaly or cerebellar ataxia. Furthermore, DNA repair defects and DNA damage in mature neurons lead to progressive cognitive impairment, which might be a common feature of Alzheimer's disease, Huntington's disease, and other polyglutamine diseases. Oxidative DNA damage and altered DNA repair gene expression are observed in GABAergic neurons in schizophrenia. These findings indicate that impaired DNA repair is a common pathological endophenotype of neurological diseases, and that DNA damage might lead to diverse disease symptoms dependent on timing and the affected cell type.

  18. DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

    SciTech Connect

    Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

    2007-12-01

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  19. DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

    SciTech Connect

    Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

    2008-02-21

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  20. Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data.

    PubMed

    Rathbun, Molly M; McElhoe, Jennifer A; Parson, Walther; Holland, Mitchell M

    2017-01-01

    Resolution of mitochondrial (mt) DNA heteroplasmy is now possible when applying a massively parallel sequencing (MPS) approach, including minor components down to 1%. However, reporting thresholds and interpretation criteria will need to be established for calling heteroplasmic variants that address a number of important topics, one of which is DNA damage. We assessed the impact of increasing amounts of DNA damage on the interpretation of minor component sequence variants in the mtDNA control region, including low-level mixed sites. A passive approach was used to evaluate the impact of storage conditions, and an active approach was employed to accelerate the process of hydrolytic damage (for example, replication errors associated with depurination events). The patterns of damage were compared and assessed in relation to damage typically encountered in poor quality samples. As expected, the number of miscoding lesions increased as conditions worsened. Single nucleotide polymorphisms (SNPs) associated with miscoding lesions were indistinguishable from innate heteroplasmy and were most often observed as 1-2% of the total sequencing reads. Numerous examples of miscoding lesions above 2% were identified, including two complete changes in the nucleotide sequence, presenting a challenge when assessing the placement of reporting thresholds for heteroplasmy. To mitigate the impact, replication of miscoding lesions was not observed in stored samples, and was rarely seen in data associated with accelerated hydrolysis. In addition, a significant decrease in the expected transition:transversion ratio was observed, providing a useful tool for predicting the presence of damage-induced lesions. The results of this study directly impact MPS analysis of minor sequence variants from poorly preserved DNA extracts, and when biological samples have been exposed to agents that induce DNA damage. These findings are particularly relevant to clinical and forensic investigations. Copyright

  1. Breaking the DNA damage response to improve cervical cancer treatment.

    PubMed

    Wieringa, Hylke W; van der Zee, Ate G J; de Vries, Elisabeth G E; van Vugt, Marcel A T M

    2016-01-01

    Every year, cervical cancer affects ∼500,000 women worldwide, and ∼275,000 patients die of this disease. The addition of platin-based chemotherapy to primary radiotherapy has increased 5-year survival of advanced-stage cervical cancer patients, which is, however, still only 66%. One of the factors thought to contribute to treatment failure is the ability of tumor cells to repair chemoradiotherapy-induced DNA damage. Therefore, sensitization of tumor cells for chemoradiotherapy via inhibition of the DNA damage response (DDR) as a novel strategy to improve therapy effect, is currently studied pre-clinically as well as in the clinic. Almost invariably, cervical carcinogenesis involves infection with the human papillomavirus (HPV), which inactivates part of the DNA damage response. This HPV-mediated partial inactivation of the DDR presents therapeutic targeting of the residual DDR as an interesting approach to achieve chemoradio-sensitization for cervical cancer. How the DDR can be most efficiently targeted, however, remains unclear. The fact that cisplatin and radiotherapy activate multiple signaling axes within the DDR further complicates a rational choice of therapeutic targets within the DDR. In this review, we provide an overview of the current preclinical and clinical knowledge about targeting the DDR in cervical cancer.

  2. ATM kinase: Much more than a DNA damage responsive protein.

    PubMed

    Guleria, Ayushi; Chandna, Sudhir

    2016-03-01

    ATM, mutation of which causes Ataxia telangiectasia, has emerged as a cardinal multifunctional protein kinase during past two decades as evidenced by various studies from around the globe. Further to its well established and predominant role in DNA damage response, ATM has also been understood to help in maintaining overall functional integrity of cells; since its mutation, inactivation or deficiency results in a variety of pathological manifestations besides DNA damage. These include oxidative stress, metabolic syndrome, mitochondrial dysfunction as well as neurodegeneration. Recently, high throughput screening using proteomics, metabolomics and transcriptomic studies revealed several proteins which might be acting as substrates of ATM. Studies that can help in identifying effective regulatory controls within the ATM-mediated pathways/mechanisms can help in developing better therapeutics. In fact, more in-depth understanding of ATM-dependent cellular signals could also help in the treatment of variety of other disease conditions since these pathways seem to control many critical cellular functions. In this review, we have attempted to put together a detailed yet lucid picture of the present-day understanding of ATM's role in various pathophysiological conditions involving DNA damage and beyond. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Ubiquitin-family modifications in the replication of DNA damage.

    PubMed

    Lehmann, Alan R

    2011-09-16

    The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Microvesicles Contribute to the Bystander Effect of DNA Damage.

    PubMed

    Lin, Xiaozeng; Wei, Fengxiang; Major, Pierre; Al-Nedawi, Khalid; Al Saleh, Hassan A; Tang, Damu

    2017-04-07

    Genotoxic treatments elicit DNA damage response (DDR) not only in cells that are directly exposed but also in cells that are not in the field of treatment (bystander cells), a phenomenon that is commonly referred to as the bystander effect (BE). However, mechanisms underlying the BE remain elusive. We report here that etoposide and ultraviolet (UV) exposure stimulate the production of microvesicles (MVs) in DU145 prostate cancer cells. MVs isolated from UV-treated DU145 and A431 epidermoid carcinoma cells as well as etoposide-treated DU145 cells induced phosphorylation of ataxia-telangiectasia mutated (ATM) at serine 1981 (indicative of ATM activation) and phosphorylation of histone H2AX at serine 139 (γH2AX) in naïve DU145 cells. Importantly, neutralization of MVs derived from UV-treated cells with annexin V significantly reduced the MV-associated BE activities. Etoposide and UV are known to induce DDR primarily through the ATM and ATM- and Rad3-related (ATR) pathways, respectively. In this regard, MV is likely a common source for the DNA damage-induced bystander effect. However, pre-treatment of DU145 naïve cells with an ATM (KU55933) inhibitor does not affect the BE elicited by MVs isolated from etoposide-treated cells, indicating that the BE is induced upstream of ATM actions. Taken together, we provide evidence supporting that MVs are a source of the DNA damage-induced bystander effect.

  5. Level of DNA damage in lead-exposed workers.

    PubMed

    Olewińska, Elżbieta; Kasperczyk, Aleksandra; Kapka, Lucyna; Kozłowska, Agnieszka; Pawlas, Natalia; Dobrakowski, Michał; Birkner, Ewa; Kasperczyk, Sławomir

    2010-01-01

    Lead plays a significant role in modern industry. This metal is related to a broad range of physiological, biochemical and behavioural dysfunctions. The genotoxic effects of lead have been studied both in animals and humans in in vitro systems but results were contradictory. The aim of this study was to investigate the association between DNA damage and occupational exposure to lead in workers. The study population consisted of 62 employees of metalworks exposed to lead in the southern region of Poland. The control group consisted of 26 office workers with no history of occupational exposure to lead. The concentration of lead (PbB) and zincprotoporphyrin (ZPP) in blood samples were measured. The DNA damage was analyzed in blood lymphocytes using alkaline comet assay. The level of DNA damage was determined as the percentage of DNA in the tail, tail length and tail moment. The lead exposure indicators were significantly higher in lead exposed group: PbB about 8.5 times and ZPP 3.3 times. Also, the percentage of DNA in the tail (60.3 ± 14 vs. 37.1 ± 17.6), comet tail length (86.9 ± 15.49 vs. 73.8 ± 19.12) and TM (57.8 ± 17.82 vs. 33.2 ± 19.13) were significantly higher in the study group when compared with the controls; however, the difference between the subgroups was only 5-10%. Years of lead exposure positively correlated with all comet assay parameters (R = 0.21-0.41). Both mean and current PbB and ZPP were correlated with tail DNA % and TM (R = 0.32; R = 0.33; R = 0.24; R = 0.26 and R = 0.34; R = 0.33; R = 0.28 and R = 0.28, respectively). This study shows that occupational exposure to lead is associated with DNA damage and confirmed that comet assay is a rapid, sensitive method suitable for biomonitoring studies.

  6. Prevention of oxidative DNA damage in rats by brussels sprouts.

    PubMed

    Deng, X S; Tuo, J; Poulsen, H E; Loft, S

    1998-03-01

    The alleged cancer preventive effects of cruciferous vegetables could be related to protection from mutagenic oxidative DNA damage. We have studied the effects of Brussels sprouts, some non-cruciferous vegetables and isolated glucosinolates on spontaneous and induced oxidative DNA damage in terms of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in groups of 6-8 male Wistar rats. Excess oxidative DNA damage was induced by 2-nitropropane (2-NP 100 mg/kg). Four days oral administration of 3 g of cooked Brussels sprouts homogenate reduced the spontaneous urinary 8-oxodG excretion by 31% (p<0.05) whereas raw sprouts, beans and endive (1:1), isolated indolyl glucosinolates and breakdown products had no significant effect. An aqueous extract of cooked Brussels sprouts (corresponding to 6.7 g vegetable per day for 4 days) decreased the spontaneous 8-oxodG excretion from 92 +/- 12 to 52 +/- 15 pmol/24 h (p<0.05). After 2-NP administration the 8-oxodG excretion was increased to 132 +/- 26 pmol/24 h (p<0.05) whereas pretreatment with the sprouts extract reduced this to 102 +/- 30 pmol/24 h (p<0.05). The spontaneous level of 8-oxodG in nuclear DNA from liver and bone marrow was not significantly affected by the sprouts extract whereas the level decreased by 27% in the kidney (p<0.05). In the liver 2-NP increased the 8-oxodG levels in nuclear DNA 8.7 and 3.8 times (p<0.05) 6 and 24 h after dose, respectively. The sprouts extract reduced this increase by 57% (p<0.05) at 6 h whereas there was no significant effect at 24 h. In the kidneys 2-NP increased the 8-oxodG levels 2.2 and 1.2 times (p<0.05) 6 and 24 h after dose, respectively. Pretreatment with the sprouts extract abolished these increases (p<0.05). Similarly, in the bone marrow the extract protected completely (p<0.05) against a 4.9-fold 2-NP induced increase (p<0.05) in the 8-oxodG level. These findings demonstrate that cooked Brussels sprouts contain bioactive substance(s) with a potential for reducing the physiological

  7. RAD54 forms DNA repair foci in response to DNA damage in living plant cells.

    PubMed

    Hirakawa, Takeshi; Hasegawa, Junko; White, Charles I; Matsunaga, Sachihiro

    2017-02-02

    Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double-strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA-TELANGIECTASIA MUTATED-SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co-localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.

  8. DNA polymerase β uses its lyase domain in a processive search for DNA damage.

    PubMed

    Howard, Michael J; Rodriguez, Yesenia; Wilson, Samuel H

    2017-04-20

    DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol β has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol β scans DNA using a processive hopping mechanism and has a mean search footprint of ∼24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol β. Application of our processive search assay into nucleosome core particles revealed that Pol β is not processive in the context of a nucleosome, and its single-turnover activity is reduced ∼500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol β mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol β. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  9. Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

    PubMed

    Fu, Dechen; Dudimah, Fred Duafalia; Zhang, Jun; Pickering, Anna; Paneerselvam, Jayabal; Palrasu, Manikandan; Wang, Hong; Fei, Peiwen

    2013-03-01

    How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.

  10. Silica radical-induced DNA damage and lipid peroxidation.

    PubMed Central

    Shi, X; Mao, Y; Daniel, L N; Saffiotti, U; Dalal, N S; Vallyathan, V

    1994-01-01

    In recent years, more attention has been given to the mechanism of disease induction caused by the surface properties of minerals. In this respect, specific research needs to be focused on the biologic interactions of oxygen radicals generated by mineral particles resulting in cell injury and DNA damage leading to fibrogenesis and carcinogenesis. In this investigation, we used electron spin resonance (ESR) and spin trapping to study oxygen radical generation from aqueous suspensions of freshly fractured crystalline silica. Hydroxyl radical (.OH), superoxide radical (O2.-) and singlet oxygen (1O2) were all detected. Superoxide dismutase (SOD) partially inhibited .OH yield, whereas catalase abolished .OH generation. H2O2 enhanced .OH generation while deferoxamine inhibited it, indicating that .OH is generated via a Haber-Weiss type reaction. These spin trapping measurements provide the first evidence that aqueous suspensions of silica particles generate O2.- and 1O2. Oxygen consumption measurements indicate that freshly fractured silica uses molecular oxygen to generate O2.- and 1O2. Electrophoretic assays of in vitro DNA strand breakages showed that freshly fractured silica induced DNA strand breakage, which was inhibited by catalase and enhanced by H2O2. In an argon atmosphere, DNA damage was suppressed, showing that molecular oxygen is required for the silica-induced DNA damage. Incubation of freshly fractured silica with linoleic acid generated linoleic acid-derived free radicals and caused dose-dependent lipid peroxidation as measured by ESR spin trapping and malondialdehyde formation. SOD, catalase, and sodium benzoate inhibited lipid peroxidation by 49, 52, and 75%, respectively, again showing the role of oxygen radicals in silica-induced lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 7. PMID:7705289

  11. DNA damage in workers occupationally exposed to pesticide mixtures.

    PubMed

    Simoniello, M F; Kleinsorge, E C; Scagnetti, J A; Grigolato, R A; Poletta, G L; Carballo, M A

    2008-11-01

    Pesticides are used in agriculture to protect crops but represent at the same time a potential risk to farmers and environment. The aim of this work is the evaluation of 54 subjects occupationally exposed to pesticides and 30 subjects as a control group using the quantification of DNA damage level by means of the alkaline Comet assay and the evaluation of repair processes. Damage index Comet assay (DICA) and damage index repair assay (DIRA) were studied in 27 pesticide applicator workers, 27 non-pesticide applicators and controls. Our results show that both exposed groups revealed significant increase in DICA when compared with controls (P < 0.0001), as well as in DIRA (P < 0.0001). However, the spraying group exhibited a marginally significant difference in DICA (P = 0.05) when years of exposure are considered and a significant difference (P < 0.05) when the personal protective equipment used by individuals was taken as a comparison factor. The influence of confounding factors on the genotoxic effects of occupational exposure to pesticides was investigated and no significant differences were observed considering age, gender, smoking and alcohol consumption in relation to DICA and DIRA. Since DNA damage is an important step in events leading from carcinogen exposure to cancer disease, our study highlights the potential health risk associated with agrochemical exposure in developing countries with vast cultivated areas, such as Argentina.

  12. The Intra-S Checkpoint Responses to DNA Damage

    PubMed Central

    Iyer, Divya Ramalingam; Rhind, Nicholas

    2017-01-01

    Faithful duplication of the genome is a challenge because DNA is susceptible to damage by a number of intrinsic and extrinsic genotoxins, such as free radicals and UV light. Cells activate the intra-S checkpoint in response to damage during S phase to protect genomic integrity and ensure replication fidelity. The checkpoint prevents genomic instability mainly by regulating origin firing, fork progression, and transcription of G1/S genes in response to DNA damage. Several studies hint that regulation of forks is perhaps the most critical function of the intra-S checkpoint. However, the exact role of the checkpoint at replication forks has remained elusive and controversial. Is the checkpoint required for fork stability, or fork restart, or to prevent fork reversal or fork collapse, or activate repair at replication forks? What are the factors that the checkpoint targets at stalled replication forks? In this review, we will discuss the various pathways activated by the intra-S checkpoint in response to damage to prevent genomic instability. PMID:28218681

  13. Environmental car exhaust pollution damages human sperm chromatin and DNA.

    PubMed

    Calogero, A E; La Vignera, S; Condorelli, R A; Perdichizzi, A; Valenti, D; Asero, P; Carbone, U; Boggia, B; De Rosa, N; Lombardi, G; D'Agata, R; Vicari, L O; Vicari, E; De Rosa, M

    2011-06-01

    The adverse role of traffic pollutants on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled. All of them were interviewed about their lifestyle. Hormone, semen samples, and environmental and biological markers of pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. LH, FSH, and testosterone serum levels were within the normal range in tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were significantly lower in these men compared with controls. Motorway tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a late sign of apoptosis, compared with controls. A significant direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship. This study showed that car exhaust exposure has a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance of the men exposed, but also for the offspring health.

  14. DNA damage and repair in telomeres: relation to aging.

    PubMed Central

    Kruk, P A; Rampino, N J; Bohr, V A

    1995-01-01

    We have established a method for the detection of DNA damage and its repair in human telomeres, the natural ends of chromosomes which are necessary for replication and critical for chromosomal stability. We find that ultraviolet light-induced pyrimidine dimers in telomeric DNA are repaired less efficiently than endogenous genes but more efficiently than inactive, noncoding regions. We have also measured telomeric length, telomeric DNA damage, and its repair in relation to the progression of aging. Telomeres are shorter in fibroblasts from an old donor compared to fibroblasts from a young donor, shortest in cells from a patient with the progeroid disorder Werner syndrome, and relatively long in fibroblasts from a patient with Alzheimer disease. Telomeric DNA repair efficiency is lower in cells from an old donor than in cells from a young donor, normal in Alzheimer cells, and slightly lower in Werner cells. It is possible that this decline in telomeric repair with aging is of functional significance to an age-related decline in genomic stability. Images Fig. 1 Fig. 2 PMID:7816828

  15. DNA-damaging activity of patulin in Escherichia coli.

    PubMed Central

    Lee, K S; Röschenthaler, R J

    1986-01-01

    At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity. PMID:2431653

  16. SUMO-mediated regulation of DNA damage repair and responses

    PubMed Central

    Sarangi, Prabha; Zhao, Xiaolan

    2015-01-01

    Sumoylation plays important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such “on-site modification” can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multi-level coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance. PMID:25778614

  17. HSV-I and the cellular DNA damage response.

    PubMed

    Smith, Samantha; Weller, Sandra K

    2015-04-01

    Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al. that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

  18. Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

    PubMed

    Schellenberg, Matthew J; Perera, Lalith; Strom, Christina N; Waters, Crystal A; Monian, Brinda; Appel, C Denise; Vilas, Caroline K; Williams, Jason G; Ramsden, Dale A; Williams, R Scott

    2016-05-05

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.

  19. Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

    PubMed

    Stephenson, Anthony A; Taggart, David J; Suo, Zucai

    2017-04-13

    Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

  20. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  1. Novobiocin Inhibits the Antimicrobial Resistance Acquired through DNA Damage-Induced Mutagenesis in Acinetobacter baumannii

    PubMed Central

    Jara, Luis M.; Pérez-Varela, María; Corral, Jordi; Arch, Marta; Cortés, Pilar; Bou, Germán; Barbé, Jordi

    2015-01-01

    Acinetobacter baumannii, a worldwide emerging nosocomial pathogen, acquires antimicrobial resistances in response to DNA-damaging agents, which increase the expression of multiple error-prone DNA polymerase components. Here we show that the aminocoumarin novobiocin, which inhibits the DNA damage response in Gram-positive bacteria, also inhibits the expression of error-prone DNA polymerases in this Gram-negative multidrug-resistant pathogen and, consequently, its potential acquisition of antimicrobial resistance through DNA damage-induced mutagenesis. PMID:26503651

  2. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  3. Oxidatively generated DNA/RNA damage in psychological stress states.

    PubMed

    Jørgensen, Anders

    2013-07-01

    Both non-pathological psychological stress states and mental disorders are associated with molecular, cellular and epidemiological signs of accelerated aging. Oxidative stress on nucleic acids is a critical component of cellular and organismal aging, and a suggested pathogenic mechanism in several age-related somatic disorders. The overall aim of the PhD project was to investigate the relation between psychopathology, psychological stress, stress hormone secretion and oxidatively generated DNA and RNA damage, as measured by the urinary excretion of markers of whole-body DNA/RNA oxidation (8-oxodG and 8-oxoGuo, respectively). The main hypothesis was that psychological stress states are associated with increased DNA/RNA damage from oxidation. In a study of 40 schizophrenia patients and 40 healthy controls matched for age and gender, we found that 8-oxodG/8-oxoGuo excretion was increased in schizophrenia patients, providing a possible molecular link between schizophrenia and its associated signs of accelerated aging. We found no association between psychopathology, perceived stress or cortisol secretion and 8-oxodG/8-oxoGuo excretion in the patients. In the controls, there were positive correlations between 8-oxodG/8-ocoGuo excretion and 9AM plasma cortisol, but no associations to perceived stress. In an animal study of experimentally induced chronic stress performed in metabolism cages, we found no increase in urinary 8-oxodG/8-oxoGuo or cerebral (hippocampal and frontal cortex) levels of oxidatively generated nucleic acid damage. However, there was a trend towards an increased expression of genes involved in DNA repair, possibly reflecting a compensatory mechanism. In a study of 220 elderly, mostly healthy individuals from the Italian InChianti cohort, we found a significant association between the 24 h urinary cortisol excretion and the excretion of 8-oxodG/8-oxoGuo, determined in the same samples. Collectively, the studies could not confirm an association between

  4. Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage.

    PubMed

    Ding, Ning; Bonham, Emily M; Hannon, Brooke E; Amick, Thomas R; Baylin, Stephen B; O'Hagan, Heather M

    2016-06-01

    At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein-protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  5. Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy

    PubMed Central

    Xu, Yang; Her, Chengtao

    2015-01-01

    Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements—ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy. PMID:26287259

  6. Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine.

    PubMed Central

    Fraga, C G; Shigenaga, M K; Park, J W; Degan, P; Ames, B N

    1990-01-01

    Oxidative damage to DNA is shown to be extensive and could be a major cause of the physiological changes associated with aging and the degenerative diseases related to aging such as cancer. The oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (oh8dG), one of the approximately 20 known oxidative DNA damage products, has been measured in DNA isolated from various organs of Fischer 344 rats of different ages. oh8dG was present in the DNA isolated from all the organs studied: liver, brain, kidney, intestine, and testes. Steady-state levels of oh8dG ranged from 8 to 73 residues per 10(6) deoxyguanosine residues or 0.2-2.0 x 10(5) residues per cell. Levels of oh8dG in DNA increased with age in liver, kidney, and intestine but remained unchanged in brain and testes. The urinary excretion of oh8dG, which presumably reflects its repair from DNA by nuclease activity, decreased with age from 481 to 165 pmol per kg of body weight per day for urine obtained from 2-month- and 25-month-old rats, respectively. 8-Hydroxyguanine, the proposed repair product of a glycosylase activity, was also assayed in the urine. We estimate approximately 9 x 10(4) oxidative hits to DNA per cell per day in the rat. The results suggest that the age-dependent accumulation of oh8dG residues observed in DNA from liver, kidney, and intestine is principally due to the slow loss of DNA nuclease activity; however, an increase in the rate of oxidative DNA damage cannot be ruled out. PMID:2352934

  7. DNA damaging potential of photoactivated p25 titanium dioxide nanoparticles.

    PubMed

    Petersen, Elijah J; Reipa, Vytas; Watson, Stephanie S; Stanley, Deborah L; Rabb, Savelas A; Nelson, Bryant C

    2014-10-20

    Titanium dioxide nanoparticles (TiO2 NPs) are found in numerous commercial and personal care products. Thus, it is necessary to understand and characterize their potential environmental health and safety risks. It is well-known that photoactivated TiO2 NPs in aerated aqueous solutions can generate highly reactive hydroxyl radicals ((•)OH), which can damage DNA. Surprisingly, recent in vitro studies utilizing the comet assay have shown that nonphotoactivated TiO2 NPs kept in the dark can also induce DNA damage. In this work, we utilize stable isotope-dilution gas chromatography/tandem mass spectrometry to quantitatively characterize the levels and types of oxidatively generated base lesions in genomic DNA exposed to NIST Standard Reference Material TiO2 NPs (Degussa P25) under precisely controlled illumination conditions. We show that DNA samples incubated in the dark for 24 h with TiO2 NPs (0.5-50 μg/mL) do not lead to the formation of base lesions. However, when the same DNA is exposed to either visible light from 400 to 800 nm (energy dose of ∼14.5 kJ/m(2)) for 24 h or UVA light at 370 nm for 30 min (energy dose of ∼10 kJ/m(2)), there is a significant formation of lesions at the 50 μg/mL dose for the visible light exposure and a significant formation of lesions at the 5 and 50 μg/mL doses for the UVA light exposure. These findings suggest that commercial P25 TiO2 NPs do not have an inherent capacity to oxidatively damage DNA bases in the absence of sufficient photoactivation; however, TiO2 NPs exposed to electromagnetic radiation within the visible portion of the light spectrum can induce the formation of DNA lesions. On the basis of these findings, comet assay processing of cells exposed to TiO2 should be performed in the dark to minimize potential artifacts from laboratory light.

  8. Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage.

    PubMed

    Raschke, Sascha; Guan, Jun; Iliakis, George

    2009-01-01

    Sucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size macromolecules is layered on the surface of a gradient whose density increases linearly from top to bottom. During centrifugation, different size macromolecules sediment through the gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the macromolecules, as well as on the density and viscosity of the gradient. In this way, macromolecules are separated by size with larger ones sedimenting towards the bottom and lighter ones remaining close to the top of the gradient. The method has been particularly successful in the size fractionation of large DNA molecules and has been extensively used to measure induction and repair of DNA breaks after exposure to clastogenic factors. Here, we describe an adaptation of this method that can be used in the analysis of newly synthesized DNA formed during DNA replication. Through size analysis of nascent DNA in alkaline sucrose gradients, variations in replication activity can be measured after exposure of cells to DNA-damaging agents. The method is particularly useful as it allows distinction between DNA damage-mediated effects on chain elongation vs. replicon initiation, which is essential for an in-depth analysis of the intra-S-phase checkpoint. This ability makes the technique unique and justifies its somewhat labour-intensive nature.

  9. Capturing snapshots of APE1 processing DNA damage

    SciTech Connect

    Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

    2015-10-12

    DNA apurinic-apyrimidinic (AP) sites are prevalent noncoding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA-repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive, owing in part to limited structural information. Here we report multiple high-resolution human APE1-DNA structures that divulge new features of the APE1 reaction, including the metal-binding site, the nucleophile and the arginine clamps that mediate product release. We also report APE1-DNA structures with a T-G mismatch 5' to the AP site, representing a clustered lesion occurring in methylated CpG dinucleotides. Moreover, these structures reveal that APE1 molds the T-G mismatch into a unique Watson-Crick-like geometry that distorts the active site, thus reducing incision. Finally, these snapshots provide mechanistic clarity for APE1 while affording a rational framework to manipulate biological responses to DNA damage.

  10. Capturing Snapshots of APE1 Processing DNA Damage

    PubMed Central

    Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

    2015-01-01

    DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

  11. Capturing snapshots of APE1 processing DNA damage

    DOE PAGES

    Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; ...

    2015-10-12

    DNA apurinic-apyrimidinic (AP) sites are prevalent noncoding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA-repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive, owing in part to limited structural information. Here we report multiple high-resolution human APE1-DNA structures that divulge new features of the APE1 reaction, including the metal-binding site, the nucleophile and the arginine clamps that mediate product release. We also report APE1-DNA structures with a T-G mismatch 5' to the AP site, representing a clustered lesion occurring in methylatedmore » CpG dinucleotides. Moreover, these structures reveal that APE1 molds the T-G mismatch into a unique Watson-Crick-like geometry that distorts the active site, thus reducing incision. Finally, these snapshots provide mechanistic clarity for APE1 while affording a rational framework to manipulate biological responses to DNA damage.« less

  12. DNA damage response in peripheral nervous system: coping with cancer therapy-induced DNA lesions.

    PubMed

    Englander, Ella W

    2013-08-01

    In the absence of blood brain barrier (BBB) the DNA of peripheral nervous system (PNS) neurons is exposed to a broader spectrum of endogenous and exogenous threats compared to that of the central nervous system (CNS). Hence, while CNS and PNS neurons cope with many similar challenges inherent to their high oxygen consumption and vigorous metabolism, PNS neurons are also exposed to circulating toxins and inflammatory mediators due to relative permeability of PNS blood nerve barrier (BNB). Consequently, genomes of PNS neurons incur greater damage and the question awaiting investigation is whether specialized repair mechanisms for maintenance of DNA integrity have evolved to meet the additional needs of PNS neurons. Here, I review data showing how PNS neurons manage collateral DNA damage incurred in the course of different anti-cancer treatments designed to block DNA replication in proliferating tumor cells. Importantly, while PNS neurotoxicity and concomitant chemotherapy-induced peripheral neuropathy (CIPN) are among major dose limiting barriers in achieving therapy goals, CIPN is partially reversible during post-treatment nerve recovery. Clearly, cell recovery necessitates mobilization of the DNA damage response and underscores the need for systematic investigation of the scope of DNA repair capacities in the PNS to help predict post-treatment risks to recovering neurons.

  13. Radiation track, DNA damage and response—a review

    NASA Astrophysics Data System (ADS)

    Nikjoo, H.; Emfietzoglou, D.; Liamsuwan, T.; Taleei, R.; Liljequist, D.; Uehara, S.

    2016-11-01

    The purpose of this paper has been to review the current status and progress of the field of radiation biophysics, and draw attention to the fact that physics, in general, and radiation physics in particular, with the aid of mathematical modeling, can help elucidate biological mechanisms and cancer therapies. We hypothesize that concepts of condensed-matter physics along with the new genomic knowledge and technologies and mechanistic mathematical modeling in conjunction with advances in experimental DNA (Deoxyrinonucleic acid molecule) repair and cell signaling have now provided us with unprecedented opportunities in radiation biophysics to address problems in targeted cancer therapy, and genetic risk estimation in humans. Obviously, one is not dealing with ‘low-hanging fruit’, but it will be a major scientific achievement if it becomes possible to state, in another decade or so, that we can link mechanistically the stages between the initial radiation-induced DNA damage; in particular, at doses of radiation less than 2 Gy and with structural changes in genomic DNA as a precursor to cell inactivation and/or mutations leading to genetic diseases. The paper presents recent development in the physics of radiation track structure contained in the computer code system KURBUC, in particular for low-energy electrons in the condensed phase of water for which we provide a comprehensive discussion of the dielectric response function approach. The state-of-the-art in the simulation of proton and carbon ion tracks in the Bragg peak region is also presented. The paper presents a critical discussion of the models used for elastic scattering, and the validity of the trajectory approach in low-electron transport. Brief discussions of mechanistic and quantitative aspects of microdosimetry, DNA damage and DNA repair are also included as developed by the authors’ work.

  14. Pathophysiology of Bronchoconstriction: Role of Oxidatively Damaged DNA Repair

    PubMed Central

    Bacsi, Attila; Pan, Lang; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Purpose of review To provide an overview on the present understanding of roles of oxidative DNA damage repair in cell signaling underlying bronchoconstriction common to, but not restricted to various forms of asthma and chronic obstructive pulmonary disease Recent findings Bronchoconstriction is a tightening of smooth muscle surrounding the bronchi and bronchioles with consequent wheezing and shortness of breath. Key stimuli include air pollutants, viral infections, allergens, thermal and osmotic changes, and shear stress of mucosal epithelium, triggering a wide range of cellular, vascular and neural events. Although activation of nerve fibers, the role of G-proteins, protein kinases and Ca++, and molecular interaction within contracting filaments of muscle are well defined, the overarching mechanisms by which a wide range of stimuli initiate these events are not fully understood. Many, if not all, stimuli increase levels of reactive oxygen species (ROS), which are signaling and oxidatively modifying macromolecules, including DNA. The primary ROS target in DNA is guanine, and 8-oxoguanine is one of the most abundant base lesions. It is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair processes. The product, free 8-oxoG base, is bound by OGG1 with high affinity, and the complex then functions as an activator of small GTPases, triggering pathways for inducing gene expression and contraction of intracellular filaments in mast and smooth muscle cells. Summary Oxidative DNA damage repair-mediated cell activation signaling result in gene expression that “primes” the mucosal epithelium and submucosal tissues to generate mediators of airway smooth muscle contractions. PMID:26694039

  15. Maintaining Genome Stability in Defiance of Mitotic DNA Damage

    PubMed Central

    Ferrari, Stefano; Gentili, Christian

    2016-01-01

    The implementation of decisions affecting cell viability and proliferation is based on prompt detection of the issue to be addressed, formulation and transmission of a correct set of instructions and fidelity in the execution of orders. While the first and the last are purely mechanical processes relying on the faithful functioning of single proteins or macromolecular complexes (sensors and effectors), information is the real cue, with signal amplitude, duration, and frequency ultimately determining the type of response. The cellular response to DNA damage is no exception to the rule. In this review article we focus on DNA damage responses in G2 and Mitosis. First, we set the stage describing mitosis and the machineries in charge of assembling the apparatus responsible for chromosome alignment and segregation as well as the inputs that control its function (checkpoints). Next, we examine the type of issues that a cell approaching mitosis might face, presenting the impact of post-translational modifications (PTMs) on the correct and timely functioning of pathways correcting errors or damage before chromosome segregation. We conclude this essay with a perspective on the current status of mitotic signaling pathway inhibitors and their potential use in cancer therapy. PMID:27493659

  16. 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

  17. Chromatin plasticity in response to DNA damage: the shape of things to come

    PubMed Central

    Polo, Sophie E.

    2016-01-01

    DNA damage poses a major threat to cell function and viability by compromising both genome and epigenome integrity. The DNA damage response indeed operates in the context of chromatin and relies on dynamic changes in chromatin organization. Here, we review the molecular bases of chromatin alterations in response to DNA damage, focusing on core histone mobilization in mammalian cells. Building on our current view of nucleosome dynamics in response to DNA damage, we highlight open challenges and avenues for future development. In particular, we discuss the different levels of regulation of chromatin plasticity during the DNA damage response and their potential impact on cell function and epigenome maintenance. PMID:25957486

  18. DNA damage in embryonic stem cells caused by nanodiamonds.

    PubMed

    Xing, Yun; Xiong, Wei; Zhu, Lin; Osawa, Eiji; Hussin, Saber; Dai, Liming

    2011-03-22

    Because of their unique photoluminescence and magnetic properties, nanodiamonds (NDs) are promising for biomedical imaging and therapeutical applications. However, these biomedical applications will hardly be realized unless the potential hazards of NDs to humans and other biological systems are ascertained. Previous studies performed in our group and others have demonstrated the excellent biocompatibility of NDs in a variety of cell lines without noticeable cytotoxicity. In the present paper, we report the first genotoxicity study on NDs. Our results showed that incubation of embryonic stem cells with NDs led to slightly increased expression of DNA repair proteins, such as p53 and MOGG-1. Oxidized nanodiamonds (O-NDs) were demonstrated to cause more DNA damage than the pristine/raw NDs (R-NDs), showing the surface chemistry specific genotoxicity. However, the DNA damages caused by either the O-NDs or the R-NDs are much less severe than those caused by multiwalled carbon nanotubes (MWNTs) observed in our previous study. These findings should have important implications for future applications of NDs in biological applications.

  19. DNA damage and mutations induced by arachidonic acid peroxidation.

    PubMed

    Lim, Punnajit; Sadre-Bazzaz, Kianoush; Shurter, Jesse; Sarasin, Alain; Termini, John

    2003-12-30

    Endogenous cellular oxidation of omega6-polyunsaturated fatty acids (PUFAs) has long been recognized as a contributing factor in the development of various cancers. The accrual of DNA damage as a result of reaction with free radical and electrophilic aldehyde products of lipid peroxidation is believed to be involved; however, the genotoxic and mutation-inducing potential of specific membrane PUFAs remains poorly defined. In the present study we have examined the ability of peroxidizing arachidonic acid (AA, 20:4omega6) to induce DNA strand breaks, base modifications, and mutations. The time-dependent induction of single-strand breaks and oxidative base modifications by AA in genomic DNA was quantified using denaturing glyoxal gel electrophoresis. Mutation spectra were determined in XP-G fibroblasts and a repair-proficient line corrected for this defect by c-DNA complementation (XP-G(+)). Mutation frequencies were elevated from approximately 5- to 30-fold over the background following reaction of DNA with AA for various times. The XPG gene product was found to be involved in the suppression of mutations after extended reaction of DNA with AA. Arachidonic acid-induced base substitutions were consistent with the presence of both oxidized and aldehyde base adducts in DNA. The frequency of multiple-base substitutions induced by AA was significantly reduced upon correction for the XPG defect (14% vs 2%, P = 0.0015). Evidence is also presented which suggests that the induced frequency of multiple mutations is lesion dependent. These results are compared to published data for mutations stimulated by alpha,beta-unsaturated aldehydes identified as products of lipid peroxidation.

  20. Activation of DNA damage response pathways as a consequence of anthracycline-DNA adduct formation.

    PubMed

    Forrest, Robert A; Swift, Lonnie P; Rephaeli, Ada; Nudelman, Abraham; Kimura, Ken-Ichi; Phillips, Don R; Cutts, Suzanne M

    2012-06-15

    The cytotoxicity of doxorubicin, a clinically used anti-neoplastic drug, can be enhanced by formaldehyde (either endogenous or exogenous) to promote the formation of doxorubicin-DNA adducts. Formaldehyde supplies the carbon required for the covalent linkage of doxorubicin to one strand of DNA, with hydrogen bonds stabilising the doxorubicin mono-adduct to the other strand of DNA, to act much like an interstrand crosslink. Interstrand crosslinks present a major challenge for cellular repair processes, requiring the activation of numerous DNA damage response proteins for resolution of the resulting DNA intermediates and damage. This work investigates DNA damage response proteins activated by doxorubicin-DNA adducts. Although p53 was phosphorylated at Serine 15 in response to adducts, long term growth inhibition of mammalian cells was not affected by p53 status. Using siRNA technology and kinase inhibitors we observed enhanced cellular sensitivity to doxorubicin-DNA adducts when the activity of the signalling protein kinases ATM and ATR were lost. Cells synchronised using a double thymidine block were sensitised to adduct-initiated cell death upon ATR knockdown, but relatively unaffected by ATM knockdown. Loss of ATR was associated with abrogation of a drug-induced G(2)/M block and induction of mitotic catastrophe, while loss of ATM was associated with drug-induced apoptosis in non-synchronised cells. These proteins may therefore be potential drug targets to achieve synergistic cytotoxic responses to doxorubicin-DNA adduct forming therapies. The analysis of these protein kinases with respect to cell cycle progression indicates that ATR is required for G(2)/M checkpoint responses while ATM appears to function in G(1) mediated responses to anthracycline adducts. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton

    SciTech Connect

    Malloy, K.D.; Holman, M.A.; Mitchell, D.

    1997-02-18

    The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlated with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. 54 refs., 4 figs., 2 tabs.

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

    PubMed

    Hamperl, Stephan; Cimprich, Karlene A

    2014-07-01

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Increased Sensitivity of DNA Damage Response-Deficient Cells to Stimulated Microgravity-Induced DNA Lesions

    PubMed Central

    Li, Nan; An, Lili; Hang, Haiying

    2015-01-01

    Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects. PMID:25915950

  4. Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

    PubMed

    Pietrzak, Monika; Halicka, H Dorota; Wieczorek, Zbigniew; Wieczorek, Jolanta; Darzynkiewicz, Zbigniew

    2008-06-01

    We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

  5. Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism

    PubMed Central

    Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

    2016-01-01

    Purpose: The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. Methods: The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2- scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays. Results: Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2- scavenging, DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Conclusion: Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3’,4’-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form. PMID:27478791

  6. 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

  7. 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.

  8. DNA-damaging effects of dental bleaching agents.

    PubMed

    Pligina, K L; Rodina, I A; Shevchenko, T V; Bekchanova, E S; Tikhonov, V P; Sirota, N P

    2012-05-01

    We studied DNA-damaging effects of dental bleaching systems containing hydrogen peroxide and/or carbamide peroxide by the "comet assay" (alkaline version). Dental bleaching systems in a hydrogen peroxide concentration range from 0.03 to 30 mM produced a genotoxic effect on isolated HeLa cells in vitro comparable with the effects of pharmacopoeial hydrogen peroxide or urea peroxide. Catalase protected the cells against products containing hydrogen peroxide and had no effect on the genotoxicity of samples containing carbamide peroxide.

  9. Are all phytochemicals useful in the preventing of DNA damage?

    PubMed

    Bacanlı, Merve; Aydın, Sevtap; Başaran, A Ahmet; Başaran, Nurşen

    2017-09-06

    Phytochemicals derived from natural plants have been used commonly for the prevention and/or treatment of different diseases due to the belief of their safety. Many plant species synthesize toxic chemicals. New natural chemicals are being discovered but their toxic effects are unknown. Phytochemicals have been regarded as possible antioxidants. But on the other hand it is suggested that various phenolic antioxidants can display pro-oxidant properties at high doses. In this review, the role of some phytochemicals (epigallocathecin gallate, carvacrol, galangin, limonene, lycopene, naringin, puerarin, terpinene, thymol and ursolic acid) on the prevention of DNA damage will be discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. DNA damage in Wistar rats exposed to dithiocarbamate pesticide mancozeb.

    PubMed

    Goldoni, A; Klauck, C R; Da Silva, S T; Da Silva, M D; Ardenghi, P G; Da Silva, L B

    2014-01-01

    Pesticides are used in large amounts in agriculture and the evaluation of their toxic effects is of major concern to public and environmental health. The aim of the present study was to investigate the genotoxic potential of a commercial formulation of the fungicide mancozeb by the micronucleus test in bone marrow and the comet assay in total blood of Wistar rats. Adult male Wistar rats were treated with a solution of mancozeb at a concentration of 40 mg/kg/day, administered intraperitoneally for 18 consecutive days, and compared to a control group. The results indicate that mancozeb induced significantly higher DNA damage as detected by the comet assay and increased the frequency of micronuclei. The results show that mancozeb is genotoxic and may adversely affect the DNA integrity of exposed organisms.

  11. DNA damage as an indicator of pollutant-induced genotoxicity

    SciTech Connect

    Shugart, L.R.

    1989-01-01

    Biological monitoring is an approach of considerable interest to scientists in the field of environmental genotoxicity who are investigating the effects of hazardous substances on the biota. In essence the technique involves an evaluation of various types of responses in living organisms for their potential to identify exposure to dangerous substances and to define or to predict subsequent deleterious effects. The rationale for the selection of DNA damage as an indicator of exposure to genotoxic agents is based mainly on the mechanisms of action of chemicals that are known mutagens and carcinogens. An alkaline unwinding assay that detects excess strand breakage within the DNA polymer was applied to sunfish in a local stream as a biological monitor for environmental genotoxicity due to industrial pollution. The study was conducted over a period of 15 months and the temporal and spatial aspects of the data were evaluated for the effect of remedial action. 16 refs., 4 figs., 4 tabs.

  12. Lymphocyte DNA damage in elevator manufacturing workers in Guangzhou, China.

    PubMed

    Lam, Tai Hing; Zhu, Chang Qi; Jiang, Chao Qiang

    2002-03-25

    To study the effect of smoking, passive smoking, alcohol drinking, and occupational exposure to low level of benzene on DNA strand breaks in elevator manufacturing workers in Guangzhou, China. Three hundred and fifty-nine workers (252 men and 107 women) of a modern elevator manufacturing factory, 205 were from production departments and 154 from managerial department. Information on the workers' health conditions, smoking, passive smoking, alcohol consumption and occupational exposure history was collected by personal interview. Lymphocyte DNA damage was measured by the Comet assay. None of the women smoked and 20.6% of the men were daily smokers. In non-smokers, the prevalence of passive smoking at work was 25% for men and 11.2% for women, and at home, 37.8 and 48.6%, respectively. Smoking significantly increased tail moment (P<0.001). Daily smokers had the largest tail moment (geometric mean, 95% CI) (0.93 microm (0.81-0.94)), followed by occasional smokers (0.76 microm (0.59-0.95)), ex-smokers (0.70 microm (0.58-0.85)), and never smokers (0.56 microm (0.53-0.60)). Tail moment increased significantly with daily tobacco consumption (cigarettes per day) (r=0.26, P<0.001) after adjusting for age, gender, occupational exposure, passive smoking, and drinking. Analysis of covariance (ANCOVA) showed that smoking (P<0.001), passive smoking at home (P=0.026), occupational exposure (P<0.001), male gender (P<0.001), and age (P=0.001) had independent effects on tail moment, whereas passive smoking at work and alcohol drinking had no significant effect. Smoking, passive smoking at home, male