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Sample records for activate dna damage

  1. Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA damage.

    PubMed

    Midorikawa, Kaoru; Uchida, Takafumi; Okamoto, Yoshinori; Toda, Chitose; Sakai, Yoshie; Ueda, Koji; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke; Kojima, Nakao

    2004-12-01

    Ethylbenzene is carcinogenic to rats and mice, while it has no mutagenic activity. We have investigated whether ethylbenzene undergoes metabolic activation, leading to DNA damage. Ethylbenzene was metabolized to 1-phenylethanol, acetophenone, 2-ethylphenol and 4-ethylphenol by rat liver microsomes. Furthermore, 2-ethylphenol and 4-ethylphenol were metabolically transformed to ring-dihydroxylated metabolites such as ethylhydroquinone and 4-ethylcatechol, respectively. Experiment with 32P-labeled DNA fragment revealed that both ethylhydroquinone and 4-ethylcatechol caused DNA damage in the presence of Cu(II). These dihydroxylated compounds also induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Catalase, methional and Cu(I)-specific chelator, bathocuproine, significantly (P<0.05) inhibited oxidative DNA damage, whereas free hydroxyl radical scavenger and superoxide dismutase did not. These results suggest that Cu(I) and H2O2 produced via oxidation of ethylhydroquinone and 4-ethylcatechol are involved in oxidative DNA damage. Addition of an endogenous reductant NADH dramatically enhanced 4-ethylcatechol-induced oxidative DNA damage, whereas ethylhydroquinone-induced DNA damage was slightly enhanced. Enhancing effect of NADH on oxidative DNA damage by 4-ethylcatechol may be explained by assuming that reactive species are generated from the redox cycle. In conclusion, these active dihydroxylated metabolites would be involved in the mechanism of carcinogenesis by ethylbenzene. PMID:15560893

  2. 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

  3. 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

  4. 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.

  5. Viral and Cellular Genomes Activate Distinct DNA Damage Responses

    PubMed Central

    Shah, Govind A.; O’Shea, Clodagh C.

    2015-01-01

    Summary In response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate ‘self’ and ‘non-self’ genomes and determine if a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability. PMID:26317467

  6. DNA binding, antioxidant activity, and DNA damage protection of chiral macrocyclic Mn(III) salen complexes.

    PubMed

    Pandya, Nirali; Khan, Noor-ul H; Prathap, K Jeya; Kureshy, Rukhsana I; Abdi, Sayed H R; Mishra, Sandhya; Bajaj, Hari C

    2012-12-01

    We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral complexes showed ability to bind with DNA, where complex S-1 exhibits the highest DNA binding constant 1.20 × 10(6) M(-1). All the compounds were screened for superoxide and hydroxyl radical scavenging activities; among them, complex S-1 exhibited significant activity with IC(50) 1.36 and 2.37 μM, respectively. Further, comet assay was used to evaluate the DNA damage protection in white blood cells against the reactive oxygen species wherein complex S-1 was found effective in protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage.

  7. Chk2 Activation Dependence on Nbs1 after DNA Damage

    PubMed Central

    Buscemi, Giacomo; Savio, Camilla; Zannini, Laura; Miccichè, Francesca; Masnada, Debora; Nakanishi, Makoto; Tauchi, Hiroshi; Komatsu, Kenshi; Mizutani, Shuki; Khanna, KumKum; Chen, Phil; Concannon, Patrick; Chessa, Luciana; Delia, Domenico

    2001-01-01

    The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G1 arrest. Here we show that the ATM-dependent activation of Chk2 by γ- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2. PMID:11438675

  8. 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

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

    PubMed Central

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

    2016-01-01

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

  10. The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome.

    PubMed

    Grenier, Lisanne; Robaire, Bernard; Hales, Barbara F

    2012-06-01

    Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence. PMID:22454429

  11. DNA-dependent protein kinase and checkpoint kinase 2 synergistically activate a latent population of p53 upon DNA damage.

    PubMed

    Jack, Melissa T; Woo, Richard A; Motoyama, Noboru; Takai, Hitoyuki; Lee, Patrick W K

    2004-04-01

    The role of the checkpoint kinase 2 (Chk2) as an upstream activator of p53 following DNA damage has been controversial. We have recently shown that Chk2 and the DNA-dependent protein kinase (DNA-PK) are both involved in DNA damage-induced apoptosis but not G(1) arrest in mouse embryo fibroblasts. Here we demonstrate that Chk2 is required to activate p53 in vitro as measured by its ability to bind its consensus DNA target sequence following DNA damage and is in fact the previously unidentified factor working synergistically with DNA-PK to activate p53. The gene mutated in ataxia telangiectasia is not involved in this p53 activation. Using wortmannin, serine 15 mutants of p53, DNA-PK null cells and Chk2 null cells, we demonstrate that DNA-PK and Chk2 act independently and sequentially on p53. Furthermore, the p53 target of these two kinases represents a latent (preexisting) population of p53. Taken together, the results from these studies are consistent with a model in which DNA damage causes an immediate and sequential modification of latent p53 by DNA-PK and Chk2, which under appropriate conditions can lead to apoptosis. PMID:14752107

  12. p53 activates G₁ checkpoint following DNA damage by doxorubicin during transient mitotic arrest.

    PubMed

    Hyun, Sun-Yi; Jang, Young-Joo

    2015-03-10

    Recovery from DNA damage is critical for cell survival. The serious damage is not able to be repaired during checkpoint and finally induces cell death to prevent abnormal cell growth. In this study, we demonstrated that 8N-DNA contents are accumulated via re-replication during prolonged recovery period containing serious DNA damage in mitotic cells. During the incubation for recovery, a mitotic delay and initiation of an abnormal interphase without cytokinesis were detected. Whereas a failure of cytokinesis occurred in cells with no relation with p53/p21, re-replication is an anomalous phenomenon in the mitotic DNA damage response in p53/p21 negative cells. Cells with wild-type p53 are accumulated just prior to the initiation of DNA replication through a G₁ checkpoint after mitotic DNA damage, even though p53 does not interrupt pre-RC assembly. Finally, these cells undergo cell death by apoptosis. These data suggest that p53 activates G₁ checkpoint in response to mitotic DNA damage. Without p53, cells with mitotic DNA damage undergo re-replication leading to accumulation of damage.

  13. Combined activation of methyl paraben by light irradiation and esterase metabolism toward oxidative DNA damage.

    PubMed

    Okamoto, Yoshinori; Hayashi, Tomohiro; Matsunami, Shinpei; Ueda, Koji; Kojima, Nakao

    2008-08-01

    Methyl paraben (MP) is often used as a preservative in foods, drugs, and cosmetics because of its high reliability in safety based on the rapid excretion and nonaccumulation following administration. Light irradiation sometimes produces unexpected activity from chemicals such as MP; furthermore, there is ample opportunity for MP to be exposed to sunlight. Here, we investigated whether MP shows DNA damage after sunlight irradiation. Two major photoproducts, p-hydroxybenzoic acid (PHBA) and 3-hydroxy methyl paraben (MP-3OH), were detected after sunlight irradiation to an aqueous MP solution. Both photoproducts were inactive in the in vitro DNA damage assay that measures oxidized guanine formed in calf thymus DNA in the presence of divalent copper ion, a known mediator of oxidative DNA damage. Simulated MP metabolism using dermal tissues after light irradiation produced these two photoproducts, which reacted with a microsomal fraction (S9) of the skin. A metabolite from MP-3OH, not PHBA, caused distinct DNA damage in the in vitro assay. This active metabolite was identified as protocatechuic acid, a hydrolyzed MP-3OH product. In addition, NADH, a cellular reductant, enhanced DNA damage by approximately five times. These results suggest that reactive oxygen species generated by the redox cycle via metal ion and catechol autoxidation are participating in oxidative DNA damage. This study reveals that MP might cause skin damage involving carcinogenesis through the combined activation of sunlight irradiation and skin esterases.

  14. Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death

    PubMed Central

    Bitomsky, Nadja; Conrad, Elisa; Moritz, Christian; Polonio-Vallon, Tilman; Sombroek, Dirk; Schultheiss, Kathrin; Glas, Carolina; Greiner, Vera; Herbel, Christoph; Mantovani, Fiamma; del Sal, Giannino; Peri, Francesca; Hofmann, Thomas G.

    2013-01-01

    Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1–HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis. PMID:24145406

  15. Lack of DNA-damaging activity of five non-nutritive sweeteners in the rat hepatocyte/DNA repair assay.

    PubMed

    Jeffrey, A M; Williams, G M

    2000-04-01

    The non-nutritive sweeteners acesulfame-K, aspartame, cyclamate, saccharin and sucralose were tested for DNA damaging activity in the rat hepatocyte/DNA repair assay. Using hepatocytes from F344 and Sprague-Dawley male rats, all were inactive despite strong responses for the positive control, 2-aminofluorene.

  16. DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation.

    PubMed

    Yi, Juan; Lu, Guang; Li, Li; Wang, Xiaozhen; Cao, Li; Lin, Ming; Zhang, Sha; Shao, Genze

    2015-05-19

    The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

  17. Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response.

    PubMed

    Wang, Yucai; Han, Xiao; Wu, Fangming; Leung, Justin W; Lowery, Megan G; Do, Huong; Chen, Junjie; Shi, Chaowei; Tian, Changlin; Li, Lei; Gong, Weimin

    2013-10-01

    The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation. PMID:23999858

  18. Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia – breast cancer susceptibility (FA-BRCA) DNA damage response network

    PubMed Central

    Abraham, Jessy; Balbo, Silvia; Crabb, David; Brooks, P.J.

    2011-01-01

    Background We recently reported that exposure of human cells in vitro to acetaldehyde resulted in activation of the Fanconi anemia-breast cancer associated (FA-BRCA) DNA damage response network. Methods To determine whether intracellular generation of acetaldehyde from ethanol metabolism can cause DNA damage and activate the FA-BRCA network, we engineered HeLa cells to metabolize alcohol by expression of human alcohol dehydrogenase 1B. Results Incubation of HeLa-ADH1B cells with ethanol (20 mM) resulted in acetaldehyde accumulation in the media which was prevented by co-incubation with 4-methyl pyrazole (4-MP), a specific inhibitor of ADH. Ethanol treatment of HeLa-ADH1B cells produced a 4-fold increase in the acetaldehyde-DNA adduct, N2-ethylidene-dGuo, and also resulted in activation of the Fanconi anemia -breast cancer susceptibility (FA-BRCA) DNA damage response network, as indicated by a monoubiquitination of FANCD2, and phosphorylation of BRCA1. Ser 1524 was identified as one site of BRCA1 phosphorylation. The increased levels of DNA adducts, FANCD2 monoubiquitination, and BRCA1 phosphorylation were all blocked by 4-MP, indicating that acetaldehyde, rather than ethanol itself, was responsible for all three responses. Importantly, the ethanol concentration we used is within the range that can be attained in the human body during social drinking. Conclusions Our results indicate that intracellular metabolism of ethanol to acetaldehyde results in DNA damage which activates the FA-BRCA DNA damage response network. PMID:21919919

  19. N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells

    PubMed Central

    2014-01-01

    Background The DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations. Methods Immunoblot and comet assays were performed to detect DNA breaks and activation of the canonical checkpoint signalling kinases following NEU damage upto 2 hours. To investigate whether mismatch repair played a role in checkpoint activation, knock-down studies were performed while flow cytometry analysis was done to understand whether the activation of the checkpoint kinases was cell cycle phase specific. Finally, breast epithelial cells were grown as 3-dimensional spheroid cultures to study whether NEU can induce upregulation of vimentin as well as disrupt cell polarity of the breast acini, thus causing transformation of epithelial cells in culture. Results We report a novel finding that NEU causes activation of major checkpoint signalling kinases, Chk1 and Chk2. This activation is temporally controlled with Chk2 activation preceding Chk1 phosphorylation, and absence of cross talk between the two parallel signalling pathways, ATM and ATR. Damage caused by NEU leads to the temporal formation of both double strand and single strand breaks. Activation of checkpoints following NEU damage is cell cycle phase dependent wherein Chk2 is primarily activated during G2-M phase whilst in S phase, there is immediate Chk1 phosphorylation and delayed Chk2 response. Surprisingly, the mismatch repair system does not play a role in checkpoint activation, at doses and duration of NEU used in the experiments. Interestingly, NEU caused disruption of the well-formed polarised spheroid archithecture and upregulation of vimentin in three-dimensional breast acini cultures of non-malignant breast epithelial cells upon NEU

  20. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  1. Maximiscin Induces DNA Damage, Activates DNA Damage Response Pathways, and Has Selective Cytotoxic Activity against a Subtype of Triple-Negative Breast Cancer.

    PubMed

    Robles, Andrew J; Du, Lin; Cichewicz, Robert H; Mooberry, Susan L

    2016-07-22

    Triple-negative breast cancers are highly aggressive, and patients with these types of tumors have poor long-term survival. These breast cancers do not express estrogen or progesterone receptors and do not have gene amplification of human epidermal growth factor receptor 2; therefore, they do not respond to available targeted therapies. The lack of targeted therapies for triple-negative breast cancers stems from their heterogeneous nature and lack of a clear definition of driver defects. Studies have recently identified triple-negative breast cancer molecular subtypes based on gene expression profiling and representative cell lines, allowing for the identification of subtype-specific drug leads and molecular targets. We previously reported the identification of a new fungal metabolite named maximiscin (1) identified through a crowdsourcing program. New results show that 1 has selective cytotoxic efficacy against basal-like 1 MDA-MB-468 cells compared to cell lines modeling other triple-negative breast cancer molecular subtypes. This compound also exhibited antitumor efficacy in a xenograft mouse model. The mechanisms of action of 1 in MDA-MB-468 cells were investigated to identify potential molecular targets and affected pathways. Compound 1 caused accumulation of cells in the G1 phase of the cell cycle, suggesting induction of DNA damage. Indeed, treatment with 1 caused DNA double-strand breaks with concomitant activation of the DNA damage response pathways, indicated by phosphorylation of p53, Chk1, and Chk2. Collectively, these results suggest basal-like triple-negative breast cancer may be inherently sensitive to DNA-damaging agents relative to other triple-negative breast cancer subtypes. These results also demonstrate the potential of our citizen crowdsourcing program to identify new lead molecules for treating the subtypes of triple-negative breast cancer. PMID:27310425

  2. DNA damage and estrogenic activity induced by the environmental pollutant 2-nitrotoluene and its metabolite

    PubMed Central

    Watanabe, Chigusa; Egami, Takashi; Midorikawa, Kaoru; Hiraku, Yusuke; Oikawa, Shinji; Kawanishi, Shosuke

    2010-01-01

    Objectives The environmental pollutant 2-nitrotoluene (2-NO2-T) is carcinogenic and reproductively toxic in animals. In this study, we elucidated the mechanisms of its carcinogenicity and reproductive toxicity. Methods We examined DNA damage induced by 2-NO2-T and its metabolite, 2-nitrosotoluene (2-NO-T), using 32P-5′-end-labeled DNA. We measured 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA and cellular DNA in cultured human leukemia (HL-60) cells treated with 2-NO2-T and 2-NO-T. 8-Oxoguanine DNA glycosylase (OGG1) gene expression in HL-60 cells was measured by real-time polymerase chain reaction (PCR). We examined estrogenic activity using an E-screen assay and a surface plasmon resonance (SPR) sensor. Results In experiments with isolated DNA fragments, 2-NO-T induced oxidative DNA damage in the presence of Cu (II) and β-nicotinamide adenine dinucleotide disodium salt (reduced form) (NADH), while 2-NO2-T did not. 2-NO-T significantly increased levels of 8-oxodG in HL-60 cells. Real-time polymerase chain reaction (PCR) analysis revealed upregulation of OGG1 gene expression induced by 2-NO-T. An E-screen assay using the human breast cancer cell line MCF-7 revealed that 2-NO2-T induced estrogen-dependent cell proliferation. In contrast, 2-NO-T decreased the cell number and suppressed 17β-estradiol-induced cell proliferation. The data obtained with the SPR sensor using estrogen receptor α and the estrogen response element supported the results of the E-screen assay. Conclusions Oxidative DNA damage caused by 2-NO-T and estrogen-disrupting effects caused by 2-NO2-T and 2-NO-T may play a role in the reproductive toxicity and carcinogenicity of these entities. PMID:21432561

  3. 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.

  4. Variable Activation of the DNA Damage Response Pathways in Patients Undergoing SPECT Myocardial Perfusion Imaging

    PubMed Central

    Hu, Shijun; Liang, Grace; Ong, Sang-Ging; Han, Leng; Sanchez-Freire, Veronica; Lee, Andrew S.; Vasanawala, Minal; Segall, George; Wu, Joseph C.

    2015-01-01

    Background Although single photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low dose radiation exposure for cardiac patients. To determine the biological effects of low dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single cell gene expression profiling. Methods and Results Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, p53, and ATM) following SPECT. The majority of patients also had either down-regulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation also had significant up-regulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant down-regulation or no difference, suggesting that some patients may potentially be more sensitive to low dose radiation exposure. Conclusions Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression post-imaging, continued care should be taken to reduce radiation exposure to both patients and operators. PMID:25609688

  5. The antioxidant activity of sulphurous thermal water protects against oxidative DNA damage: a comet assay investigation.

    PubMed

    Braga, P C; Ceci, C; Marabini, L; Nappi, G

    2013-04-01

    Various studies have recently shown that sulphurous waters acts against the oxidants released during respiratory bursts of human neutrophils, and free radicals such as HO•, O2¯•, Tempol and Fremy's salt. However, there is still a lack of data concerning their direct protection of DNA. The aim of this study was to investigate the antigenotoxicity effects of sulphurous water, which has never been previously investigated for this purpose, using the alkaline single cell gel electrophoresis (SCGE) approach (comet assay). The comet assay is a sensitive method for assessing DNA fragmentation in individual cells in genotoxicity studies but can also be used to investigate the activity of agents that protect against DNA damage. The extent of migration was measured by means of SCGE, and DNA damage was expressed as tail moment. All of these assays were made using natural sulphurous water, degassed sulphurous water (no detectable HS), and reconstituted sulphurous water (degassed plus NaHS). DNA damages was significantly inhibited by natural water with HS concentrations of 5.0 and 2.5 μg/mL. The use of degassed water did not lead to any significant differences from baseline values, whereas the reconstituted water led to significant results overlapping those obtained using natural water. These findings confirm the importance of the presence of an HS group (reductive activity) and indicate that, in addition to their known mucolytic activity and trophic effects on respiratory mucosa, HS groups in sulphurous water also protect against oxidative DNA damage and contribute to the water's therapeutic effects on upper and lower airway inflammatory diseases.

  6. ATF2 on the Double – Activating Transcription Factor and DNA Damage Response Protein

    PubMed Central

    Bhoumik, Anindita; Bergami, Pablo Lopez; Ronai, Ze’ev

    2010-01-01

    The Activating Transcription Factor 2 (ATF2) has been implicated in transcription and DNA damage control, through its phosphorylation by JNK/p38 or ATM/ATR, respectively. ATF2 activities have also been associated with skin tumor development and progression. Here we summarize our present understanding of ATF2 regulation, function and contribution to malignant and non malignant skin tumor development. PMID:17935492

  7. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

    PubMed

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T; Passmore, Lori A; Patel, Ketan J; Olsen, Jesper V; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage.

  8. DNA damage checkpoints in mammals.

    PubMed

    Niida, Hiroyuki; Nakanishi, Makoto

    2006-01-01

    DNA damage is a common event and probably leads to mutation or deletion within chromosomal DNA, which may cause cancer or premature aging. DNA damage induces several cellular responses including DNA repair, checkpoint activity and the triggering of apoptotic pathways. DNA damage checkpoints are associated with biochemical pathways that end delay or arrest of cell-cycle progression. These checkpoints engage damage sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-RFC complex, in the detection of DNA damage and transduction of signals to ATM, ATR, Chk1 and Chk2 kinases. Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks) which inhibit cell-cycle progression. In this review, we discuss the molecular mechanisms by which DNA damage is recognized by sensor proteins and signals are transmitted to Cdks. We classify the genes involved in checkpoint signaling into four categories, namely sensors, mediators, transducers and effectors, although their proteins have the broad activity, and thus this classification is for convenience and is not definitive. PMID:16314342

  9. YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage

    PubMed Central

    Ciamporcero, Eric; Shen, He; Ramakrishnan, Swathi; Ku, Sheng Yu; Chintala, Sreenivasulu; Shen, Li; Adelaiye, Remi; Miles, Kiersten Marie; Ullio, Chiara; Pizzimenti, Stefania; Daga, Martina; Azabdaftari, Gissou; Attwood, Kris; Johnson, Candace; Zhang, Jianmin; Barrera, Giuseppina; Pili, Roberto

    2015-01-01

    Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knock-down sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC. PMID:26119935

  10. Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage.

    PubMed

    Hofstetter, Christine; Kampka, Justyna M; Huppertz, Sascha; Weber, Heike; Schlosser, Andreas; Müller, Albrecht M; Becker, Matthias

    2016-02-15

    Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2- and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs. PMID:26759175

  11. Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage.

    PubMed

    Hofstetter, Christine; Kampka, Justyna M; Huppertz, Sascha; Weber, Heike; Schlosser, Andreas; Müller, Albrecht M; Becker, Matthias

    2016-02-15

    Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2- and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.

  12. Activation of DNA Damage Response Pathways during Lytic Replication of KSHV.

    PubMed

    Hollingworth, Robert; Skalka, George L; Stewart, Grant S; Hislop, Andrew D; Blackbourn, David J; Grand, Roger J

    2015-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Human tumour viruses such as KSHV are known to interact with the DNA damage response (DDR), the molecular pathways that recognise and repair lesions in cellular DNA. Here it is demonstrated that lytic reactivation of KSHV leads to activation of the ATM and DNA-PK DDR kinases resulting in phosphorylation of multiple downstream substrates. Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release. There is no activation of the ATR-CHK1 pathway following lytic replication and CHK1 phosphorylation is inhibited at later times during the lytic cycle. Despite evidence of double-strand breaks and phosphorylation of H2AX, 53BP1 foci are not consistently observed in cells containing lytic virus although RPA32 and MRE11 localise to sites of viral DNA synthesis. Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle. KSHV appears then to selectively activate DDR pathways, modulate cell cycle progression and recruit DDR proteins to sites of viral replication during the lytic cycle.

  13. The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

    SciTech Connect

    Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

    2004-02-01

    Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

  14. Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair

    PubMed Central

    Joyce, Eric F.; Pedersen, Michael; Tiong, Stanley; White-Brown, Sanese K.; Paul, Anshu; Campbell, Shelagh D.

    2011-01-01

    Ataxia telangiectasia–mutated (ATM) and ataxia telangiectasia–related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis. PMID:22024169

  15. Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

    PubMed Central

    Kuznetsov, Nikita A.; Bergonzo, Christina; Campbell, Arthur J.; Li, Haoquan; Mechetin, Grigory V.; de los Santos, Carlos; Grollman, Arthur P.; Fedorova, Olga S.; Zharkov, Dmitry O.; Simmerling, Carlos

    2015-01-01

    Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination. PMID:25520195

  16. Regulation of zygotic genome activation and DNA damage checkpoint acquisition at the mid-blastula transition

    PubMed Central

    Zhang, Maomao; Kothari, Priyanka; Mullins, Mary; Lampson, Michael A.

    2014-01-01

    Following fertilization, oviparous embryos undergo rapid, mostly transcriptionally silent cleavage divisions until the mid-blastula transition (MBT), when large-scale developmental changes occur, including zygotic genome activation (ZGA) and cell cycle remodeling, via lengthening and checkpoint acquisition. Despite their concomitant appearance, whether these changes are co-regulated is unclear. Three models have been proposed to account for the timing of (ZGA). One model implicates a threshold nuclear to cytoplasmic (N:C) ratio, another stresses the importance cell cycle elongation, while the third model invokes a timer mechanism. We show that precocious Chk1 activity in pre-MBT zebrafish embryos elongates cleavage cycles, thereby slowing the increase in the N:C ratio. We find that cell cycle elongation does not lead to transcriptional activation. Rather, ZGA slows in parallel with the N:C ratio. We show further that the DNA damage checkpoint program is maternally supplied and independent of ZGA. Although pre-MBT embryos detect damage and activate Chk2 after induction of DNA double-strand breaks, the Chk1 arm of the DNA damage response is not activated, and the checkpoint is nonfunctional. Our results are consistent with the N:C ratio model for ZGA. Moreover, the ability of precocious Chk1 activity to delay pre-MBT cell cycles indicate that lack of Chk1 activity limits checkpoint function during cleavage cycles. We propose that Chk1 gain-of-function at the MBT underlies cell cycle remodeling, whereas ZGA is regulated independently by the N:C ratio. PMID:25558827

  17. CHK1 and RAD51 activation after DNA damage is regulated via urokinase receptor/TLR4 signaling

    PubMed Central

    Narayanaswamy, Pavan B; Tkachuk, Sergey; Haller, Hermann; Dumler, Inna; Kiyan, Yulia

    2016-01-01

    Mechanisms of DNA damage and repair signaling are not completely understood that hinder the efficiency of cancer therapy. Urokinase-type plasminogen activator receptor (PLAUR) is highly expressed in most solid cancers and serves as a marker of poor prognosis. We show that PLAUR actively promotes DNA repair in cancer cells. On the contrary, downregulation of PLAUR expression results in delayed DNA repair. We found PLAUR to be essential for activation of Checkpoint kinase 1 (CHK1); maintenance of cell cycle arrest after DNA damage in a TP53-dependent manner; expression, nuclear import and recruitment to DNA-damage foci of RAD51 recombinase, the principal protein involved in the homologous recombination repair pathway. Underlying mechanism implies auto-/paracrine signaling of PLAUR/TLR4 receptor complex leading to activation of CHK1 and DNA repair. The signaling is induced by a danger molecule released by DNA-damaged cells and mediates, at least partially, activation of DNA-damage response. This study describes a new mechanism of DNA repair activation initiated by auto-/paracrine signaling of membrane receptors PLAUR/TLR4. It adds to the understanding of role of PLAUR in cancer and provides a rationale for therapeutic targeting of PLAUR/TLR4 interaction in TP53-positive cancers. PMID:27685627

  18. Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage

    SciTech Connect

    Laha, Dipranjan; Pramanik, Arindam; Laskar, Aparna; Jana, Madhurya; Pramanik, Panchanan; Karmakar, Parimal

    2014-11-15

    Highlights: • Spherical and sheet shaped copper oxide nanoparticles were synthesized. • Physical characterizations of these nanoparticles were done by TEM, DLS, XRD, FTIR. • They showed shape dependent antibacterial activity on different bacterial strain. • They induced both membrane damage and ROS mediated DNA damage in bacteria. - Abstract: In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering. The antibacterial activity of these nanoparticles was determined on both gram positive and gram negative bacterial. Spherical shaped copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and bacterial strain.

  19. Antioxidant and DNA Damage Protecting Activity of Exopolysaccharides from the Endophytic Bacterium Bacillus cereus SZ1.

    PubMed

    Zheng, Li Ping; Zou, Tin; Ma, Yan Jun; Wang, Jian Wen; Zhang, Yu Qing

    2016-01-01

    An endophytic bacterium was isolated from the Chinese medicinal plant Artemisia annua L. The phylogenetic and physiological characterization indicated that the isolate, strain SZ-1, was Bacillus cereus. The endophyte could produce an exopolysaccharide (EPS) at 46 mg/L. The 1,1-diphenyl-2-picrylhydracyl (DPPH) radical scavenging activity of the EPS reached more than 50% at 3-5 mg/mL. The EPS was also effective in scavenging superoxide radical in a concentration dependent fashion with an EC50 value of 2.6 mg/mL. The corresponding EC50 for scavenging hydroxyl radical was 3.1 mg/mL. Moreover, phenanthroline-copper complex-mediated chemiluminescent emission of DNA damage was both inhibited and delayed by EPS. The EPS at 0.7-1.7 mg/mL also protected supercoiled DNA strands in plasmid pBR322 against scission induced by Fenton-mediated hydroxyl radical. The preincubation of PC12 cells with the EPS prior to H₂O₂ exposure increased the cell survival and glutathione (GSH) level and catalase (CAT) activities, and decreased the level of malondialdehyde (MDA) and lactate dehydrogenase (LDH) activity in a dose-dependent manner, suggesting a pronounced protective effect against H₂O₂-induced cytotoxicity. Our study indicated that the EPS could be useful for preventing oxidative DNA damage and cellular oxidation in pharmaceutical and food industries. PMID:26861269

  20. Activation of WIP1 Phosphatase by HTLV-1 Tax Mitigates the Cellular Response to DNA Damage

    PubMed Central

    Dayaram, Tajhal; Lemoine, Francene J.; Donehower, Lawrence A.; Marriott, Susan J.

    2013-01-01

    Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome. PMID:23405243

  1. DNA Damage and the Activation of the p53 Pathway Mediate Alterations in Metabolic and Secretory Functions of Adipocytes.

    PubMed

    Vergoni, Bastien; Cornejo, Pierre-Jean; Gilleron, Jérôme; Djedaini, Mansour; Ceppo, Franck; Jacquel, Arnaud; Bouget, Gwennaelle; Ginet, Clémence; Gonzalez, Teresa; Maillet, Julie; Dhennin, Véronique; Verbanck, Marie; Auberger, Patrick; Froguel, Philippe; Tanti, Jean-François; Cormont, Mireille

    2016-10-01

    Activation of the p53 pathway in adipose tissue contributes to insulin resistance associated with obesity. However, the mechanisms of p53 activation and the effect on adipocyte functions are still elusive. Here we found a higher level of DNA oxidation and a reduction in telomere length in adipose tissue of mice fed a high-fat diet and an increase in DNA damage and activation of the p53 pathway in adipocytes. Interestingly, hallmarks of chronic DNA damage are visible at the onset of obesity. Furthermore, injection of lean mice with doxorubicin, a DNA damage-inducing drug, increased the expression of chemokines in adipose tissue and promoted its infiltration by proinflammatory macrophages and neutrophils together with adipocyte insulin resistance. In vitro, DNA damage in adipocytes increased the expression of chemokines and triggered the production of chemotactic factors for macrophages and neutrophils. Insulin signaling and effect on glucose uptake and Glut4 translocation were decreased, and lipolysis was increased. These events were prevented by p53 inhibition, whereas its activation by nutlin-3 reproduced the DNA damage-induced adverse effects. This study reveals that DNA damage in obese adipocytes could trigger p53-dependent signals involved in alteration of adipocyte metabolism and secretory function leading to adipose tissue inflammation, adipocyte dysfunction, and insulin resistance. PMID:27388216

  2. miR-34 activity is modulated through 5'-end phosphorylation in response to DNA damage.

    PubMed

    Salzman, David W; Nakamura, Kotoka; Nallur, Sunitha; Dookwah, Michelle T; Metheetrairut, Chanatip; Slack, Frank J; Weidhaas, Joanne B

    2016-01-01

    MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level. PMID:26996824

  3. KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage.

    PubMed

    Gong, Eun-Yeung; Smits, Veronique A J; Fumagallo, Felipe; Piscitello, Desiree; Morrice, Nick; Freire, Raimundo; Gillespie, David A

    2015-01-01

    The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation. PMID:26039276

  4. Activation of ATM depends on chromatin interactions occurring before induction of DNA damage.

    PubMed

    Kim, Yong-Chul; Gerlitz, Gabi; Furusawa, Takashi; Catez, Frédéric; Nussenzweig, Andre; Oh, Kyu-Seon; Kraemer, Kenneth H; Shiloh, Yosef; Bustin, Michael

    2009-01-01

    Efficient and correct responses to double-stranded breaks (DSB) in chromosomal DNA are crucial for maintaining genomic stability and preventing chromosomal alterations that lead to cancer. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signalling network of the cellular response to DSB. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both before and after DSB formation, thereby optimizing its activation. Loss of HMGN1 or ablation of its ability to bind to chromatin reduces the levels of ionizing radiation (IR)-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys 14 of histone H3 (H3K14) in an HMGN1-dependent manner and treatment of cells with histone deacetylase inhibitors bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation after DNA damage. PMID:19079244

  5. Biological Clues to Potent DNA-Damaging Activities in Food and Flavoring

    PubMed Central

    Hossain, M. Zulfiquer; Gilbert, Samuel F.; Patel, Kalpesh; Ghosh, Soma; Bhunia, Anil K.; Kern, Scott E.

    2013-01-01

    Population differences in age-related diseases and cancer could stem from differences in diet. To characterize DNA strand-breaking activities in selected foods/beverages, flavorings, and some of their constituent chemicals, we used p53R cells, a cellular assay sensitive to such breaks. Substances testing positive included reference chemicals: quinacrine (peak response, 51X) and etoposide (33X); flavonoids: EGCG (19X), curcumin (12X), apigenin (9X), and quercetin (7X); beverages: chamomile (11X), green (21X), and black tea (26X) and coffee (3 to 29X); and liquid smoke (4 to 28X). Damage occurred at dietary concentrations: etoposide near 5 μg/ml produced responses similar to a 1:1000 dilution of liquid smoke, a 1:20 dilution of coffee, and a 1:5 dilution of tea. Pyrogallol-related chemicals and tannins are present in dietary sources and individually produced strong activity: pyrogallol (30X), 3-methoxycatechol (25X), gallic acid (21X), and 1,2,4-benzenetriol (21X). From structure-activity relationships, high activities depended on specific orientations of hydroxyls on the benzene ring. Responses accompanied cellular signals characteristic of DNA breaks such as H2AX phosphorylation. Breaks were also directly detected by comet assay. Cellular toxicological effects of foods and flavorings could guide epidemiologic and experimental studies of potential disease risks from DNA strand-breaking chemicals in diets. PMID:23402862

  6. Phenolic profile, antioxidant potential and DNA damage protecting activity of sugarcane (Saccharum officinarum).

    PubMed

    Abbas, Syed Rizwan; Sabir, Syed Mubashar; Ahmad, Syed Dilnawaz; Boligon, Aline Augusti; Athayde, Margareth Linde

    2014-03-15

    The present study investigated the antioxidant and phenolic composition of sugarcane. The leaves and juices of thirteen varieties of sugarcane were studied for their antioxidant activity and protective effect on DNA damage. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) assay was used to determine the radical scavenging activities in leaves and juices. Different varieties of sugarcane showed good antioxidant properties, IC50 values ranged from 20.82 to 27.47 μg/ml for leaves and from 63.95 to higher than 200 μg/ml for juice. The leaves and juice possess strong ability to protect against DNA damage induced by hydroxyl radical generated in Fenton reaction. The major phenolic acids, some flavonoid aglycone and glycosides were identified in leaves by high performance liquid chromatography. Ferulic acid (14.63 ± 0.03 mg/g), cumaric acid (11.65 ± 0.03 mg/g), quercetrin (10.96 ± 0.02 mg/g), caffeic acid (9.16 ± 0.01 mg/g) and ellagic acid (9.03 ± 0.02 mg/g) were prédominant in infusion of sugarcane.

  7. Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response

    PubMed Central

    Bujdoso, Raymond; Landgraf, Matthias; Jackson, Walker S; Thackray, Alana M

    2015-01-01

    Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease. PMID:26279981

  8. Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials of Moringa oleifera.

    PubMed

    Singh, Brahma N; Singh, B R; Singh, R L; Prakash, D; Dhakarey, R; Upadhyay, G; Singh, H B

    2009-06-01

    The aqueous extract of leaf (LE), fruit (FE) and seed (SE) of Moringa oleifera was assessed to examine the ability to inhibit the oxidative DNA damage, antioxidant and anti-quorum sensing (QS) potentials. It was found that these extracts could significantly inhibit the OH-dependent damage of pUC18 plasmid DNA and also inhibit synergistically with trolox, with an activity sequence of LE > FE > SE. HPLC and MS/MS analysis was carried out, which showed the presence of gallic acid, chlorogenic acid, ellagic acid, ferulic acid, kaempferol, quercetin and vanillin. The LE was with comparatively higher total phenolics content (105.04 mg gallic acid equivalents (GAE)/g), total flavonoids content (31.28 mg quercetin equivalents (QE)/g), and ascorbic acid content (106.95 mg/100 g) and showed better antioxidant activity (85.77%), anti-radical power (74.3), reducing power (1.1 ascorbic acid equivalents (ASE)/ml), inhibition of lipid peroxidation, protein oxidation, OH-induced deoxyribose degradation, and scavenging power of superoxide anion and nitric oxide radicals than did the FE, SE and standard alpha-tocopherol. Eventually, LE and FE were found to inhibit violacein production, a QS-regulated behavior in Chromobacterium violaceum 12472.

  9. Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro

    PubMed Central

    Hossain, M. Zulfiquer; Patel, Kalpesh; Kern, Scott E.

    2014-01-01

    Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. PMID:24842839

  10. Antioxidant activity of Coriandrum sativum and protection against DNA damage and cancer cell migration

    PubMed Central

    2013-01-01

    Background Coriandrum sativum is a popular culinary and medicinal herb of the Apiaceae family. Health promoting properties of this herb have been reported in pharmacognostical, phytochemical and pharmacological studies. However, studies on C. sativum have always focused on the aerial parts of the herb and scientific investigation on the root is limited. The aim of this research was to investigate the antioxidant and anticancer activities of C. sativum root, leaf and stem, including its effect on cancer cell migration, and its protection against DNA damage, with special focus on the roots. Methods Powdered roots, leaves and stems of C. sativum were extracted through sequential extraction using hexane, dichloromethane, ethyl acetate, methanol and water. Total phenolic content, FRAP and DPPH radical scavenging activities were measured. Anti-proliferative activitiy on the breast cancer cell line, MCF-7, was assayed using the MTT assay. Activities of the antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, and of the caspases-3, -8 and -9 were assayed on treatment with the extract. Cell cycle progression was analysed using flow cytometry. The scratch motility assay was used to assess inhibition of MCF-7 cell migration. DNA damage in 3 T3-L1 fibroblasts was evaluated by the comet assay. The components in the extract were identified by HPLC and GC-MS. Results The ethyl acetate extract of C. sativum roots showed the highest antiproliferative activity on MCF-7 cells (IC50 = 200.0 ± 2.6 μg/mL) and had the highest phenolic content, FRAP and DPPH scavenging activities among the extracts. C. sativum root inhibited DNA damage and prevented MCF-7 cell migration induced by H2O2, suggesting its potential in cancer prevention and inhibition of metastasis. The extract exhibited anticancer activity in MCF-7 cells by affecting antioxidant enzymes possibly leading to H2O2 accumulation, cell cycle arrest at the G2/M phase and apoptotic cell death by

  11. In vivo antigenotoxic activity of watercress juice (Nasturtium officinale) against induced DNA damage.

    PubMed

    Casanova, Natalia A; Ariagno, Julia I; López Nigro, Marcela M; Mendeluk, Gabriela R; de los A Gette, María; Petenatti, Elisa; Palaoro, Luis A; Carballo, Marta A

    2013-09-01

    The present study was carried out to investigate the genotoxicity as well as possible protective activity against damage induced by cyclophosphamide (CP) of the aqueous juice of watercress (Nasturtium officinale, W.T. Aiton) in vivo. Male and female Swiss mice 7-8 weeks old (N = 48) were treated by gavage with 1 g kg(-1) body weight and 0.5 g kg(-1) body weight of watercress juice during 15 consecutive days. Genotoxicity and its possible protective effect were tested by the comet assay in peripheral blood cells and the micronucleus test in bone marrow. In addition, biopsies of the bladder, epididymis and testicles of mice were performed to extend the experimental design. Watercress juice per se did not induce genetic damage according to the comet assay and micronucleus study, exhibiting a protective activity against CP (P < 0.05 and P < 0.001, respectively). The comparative analysis of bladder histological changes obtained in the watercress plus CP group against those treated with CP alone suggests a probable protective effect. Further studies are needed in order to establish the protective role of watercress juice against DNA damage.

  12. DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions.

    PubMed

    Insinga, Alessandra; Cicalese, Angelo; Faretta, Mario; Gallo, Barbara; Albano, Luisa; Ronzoni, Simona; Furia, Laura; Viale, Andrea; Pelicci, Pier Giuseppe

    2013-03-01

    DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.

  13. 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

  14. XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    PubMed Central

    Kaushik Tiwari, Meetu; Rogers, Faye A.

    2013-01-01

    DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation. PMID:23913414

  15. MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation

    PubMed Central

    Wong, Po Yee; Ma, Hoi Tang; Lee, Hyun-jung; Poon, Randy Y. C.

    2016-01-01

    The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation. PMID:26923777

  16. Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells

    PubMed Central

    Puebla-Osorio, N; Damiani, E; Bover, L; Ullrich, S E

    2015-01-01

    Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2–M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment. PMID:25950475

  17. Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage.

    PubMed

    Mahl, Camila Donato; Behling, Camile Saul; Hackenhaar, Fernanda S; de Carvalho e Silva, Mélany Natuane; Putti, Jordana; Salomon, Tiago B; Alves, Sydney Hartz; Fuentefria, Alexandre; Benfato, Mara S

    2015-07-01

    In this study, we assessed the generation of reactive oxygen species (ROS) induced by subinhibitory concentration of fluconazole in susceptible and resistant Candida glabrata strains at stationary growth phase and measured their oxidative responses parameters: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST), consumption of hydrogen peroxide, and total glutathione, as well as oxidative damage in lipids, proteins, and DNA. Data showed that fluconazole increased generation of ROS and GPx and SOD enzymatic activity in treated cells; however, these enzymatic activities did not differ between resistant and susceptible strains. Susceptible strains exhibited higher GST activity than resistant, and when susceptible cells were treated with fluconazole, GST activity decreased. Fluconazole treatment cause oxidative damage only in DNA. There are a possible participation of ROS, as organic peroxides and O2(•-), in antifungal mechanism of fluconazole, which results in higher GPx and SOD enzymatic activities and oxidative DNA damage in C. glabrata.

  18. Enhancement of anti-proliferative activities of Metformin, when combined with Celecoxib, without increasing DNA damage.

    PubMed

    Ullah, Asad; Ashraf, Muhammad; Javeed, Aqeel; Anjum, Aftab Ahmad; Attiq, Ali; Ali, Sarwat

    2016-07-01

    Pathophysiological changes in diabetes like hyperglycemia, oxidative stress, insulin resistance and compensatory hyperinsulinemia predispose cells to malignant transformation and damage DNA repair mechanism. This study was designed to explore the potential synergistic toxic effects of anti-diabetic drug (Metformin), and an analgesic drug (Celecoxib) at cellular level. MTT assay run on Vero cell line revealed that the combinations of Metformin and Celecoxib augment the anti-proliferative effects, whereas Single cell gel electrophoresis spotlighted that Metformin produce non-significant DNA damage with the threshold concentration of 400μg/ml in peripheral blood mononuclear cells (lymphocytes and monocytes), while Celecoxib produced significant (P<0.05) DNA damage (class III comets) above the concentration of 75μg/ml, however the DNA damage or DNA tail protrusions by combinations of both drugs were less than what was observed with Celecoxib alone. Metformin or Celecoxib did not appear mutagenic against any mutant strains (TA 100 and TA 98) but their combination exhibited slight mutagenicity at much higher concentration. The results obtained at concentrations higher than the therapeutic level of drugs and reflect that Metformin in combination with Celecoxib synergistically inhibits the cell proliferation in a concentration dependent pattern. Since, this increase in cytotoxicity did not confer an increase in DNA damage; this combination could be adopted to inhibit the growth of malignant cell without producing any genotoxic or mutagenic effects at cellular level. PMID:27327526

  19. Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method.

    PubMed

    Guo, Shanshan; Deng, Qianchun; Xiao, Junsong; Xie, Bijun; Sun, Zhida

    2007-04-18

    The antioxidant activities of three parts (peel, juice, and seed) and extracts of three pomegranate varieties in China were investigated by using a chemiluminescence (CL) method in vitro. The scavenging ability of pomegranate extracts (PEs) on superoxide anion, hydroxide radical, and hydrogen peroxide was determined by the pyrogallol-luminol system, the CuSO4-Phen-Vc-H2O2 system, and the luminol-H2O2 system, respectively. DNA damage preventing the effect of PE was determined by the CuSO4-Phen-Vc-H2O2-DNA CL system. The results showed that the peel extract of red pomegranate had the best effect on the scavenging ability of superoxide anion because its IC50 value (4.01 +/- 0.09 microg/mL) was the lowest in all PEs. The seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the IC50 value was 1.69 +/- 0.03 microg/mL). The peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide, which had the lowest IC50 value (0.032 +/- 0.003 microg/mL) in the nine extracts. The seed extract of white pomegranate (the IC50 value was 3.67 +/- 0.03 microg/mL) was the most powerful on the DNA damage-preventing effect in all of the PEs. Also, the statistical analysis indicated that there were significant differences (at P < 0.05) among the extracts of the different varieties and parts in each system. PMID:17381116

  20. Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance.

    PubMed

    Palle, Komaraiah; Mani, Chinnadurai; Tripathi, Kaushlendra; Athar, Mohammad

    2015-01-01

    The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for

  1. Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance

    PubMed Central

    Palle, Komaraiah; Mani, Chinnadurai; Tripathi, Kaushlendra; Athar, Mohammad

    2015-01-01

    The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for

  2. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

    PubMed

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

    2015-09-30

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

  3. Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint.

    PubMed

    Man, Wing Yu; Mak, Joyce P Y; Poon, Randy Y C

    2014-01-01

    Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2 /M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint. PMID:24238094

  4. Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint.

    PubMed

    Man, Wing Yu; Mak, Joyce P Y; Poon, Randy Y C

    2014-01-01

    Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2 /M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.

  5. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

    PubMed

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

    2015-01-01

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

  6. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells

    PubMed Central

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N.; Guo, Lei; Mei, Nan

    2015-01-01

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

  7. Sperm DNA damage and its relation with leukocyte DNA damage.

    PubMed

    Babazadeh, Zahra; Razavi, Shahnaz; Tavalaee, Marziyeh; Deemeh, Mohammad Reza; Shahidi, Maryam; Nasr-Esfahani, Mohammad Hossein

    2010-01-01

    DNA fragmentation in human sperm has been related to endogenous and exogenous factors. Exogenous factors can also affect leukocyte DNA integrity. This study evaluated the relation between sperm DNA damage and leukocyte DNA integrity, as a predictor of exogenous factors. DNA damage in the sperm and leukocytes of 41 individuals undergoing ICSI were measured by Comet assay. In addition, sperm chromatin dispersion (SCD) was carried out on semen samples. A positive correlation was observed between the DNA integrity of sperm with leukocytes. When patients were divided into low and high DNA exposure groups, sperm DNA fragmentation was significantly different between the two groups. Cleavage rate and embryo quality showed significant correlation with leukocyte DNA integrity. The results showed that leukocyte DNA integrity could be used to identify individuals at high risk in order to reduce the extent of DNA damage in patients before ICSI in order to improve the subsequent outcome of this procedure.

  8. Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity.

    PubMed

    Dickey, Jennifer S; Baird, Brandon J; Redon, Christophe E; Avdoshina, Valeriya; Palchik, Guillermo; Wu, Junfang; Kondratyev, Alexei; Bonner, William M; Martin, Olga A

    2012-11-01

    Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo.

  9. Anti-proliferative activity and chemoprotective effects towards DNA oxidative damage of fresh and cooked Brassicaceae.

    PubMed

    Ferrarini, Lisa; Pellegrini, Nicoletta; Mazzeo, Teresa; Miglio, Cristiana; Galati, Serena; Milano, Francesco; Rossi, Carlo; Buschini, Annamaria

    2012-05-01

    Epidemiological evidence shows that regular consumption of Brassicaceae is associated with a reduced risk of cancer and heart disease. Cruciferous species are usually processed before eating and the real impact of cooking practices on their bioactive properties is not fully understood. We have evaluated the effect of common cooking practices (boiling, microwaving, and steaming) on the biological activities of broccoli, cauliflower and Brussels sprouts. Anti-proliferative and chemoprotective effects towards DNA oxidative damage of fresh and cooked vegetable extracts were evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and Comet assays on HT-29 human colon carcinoma cells. The fresh vegetable extracts showed the highest anti-proliferative and antioxidant activities on HT-29 cells (broccoli>cauliflower = Brussels sprouts). No genotoxic activity was detected in any of the samples tested. The cooking methods that were applied influenced the anti-proliferative activity of Brassica extracts but did not alter considerably the antioxidant activity presented by the raw vegetables. Raw, microwaved, boiled (except broccoli) and steamed vegetable extracts, at different concentrations, presented a protective antioxidative action comparable with vitamin C (1 mm). These data provide new insight into the influence of domestic treatment on the quality of food, which could support the recent epidemiological studies suggesting that consumption of cruciferous vegetables, mainly cooked, may be related to a reduced risk of developing cancer.

  10. Soy isoflavones have antimutagenic activity on DNA damage induced by the antileishmanial Glucantime (meglumine antimoniate).

    PubMed

    Cantanhêde, Ludymila Furtado; Almeida, Laís Pinheiro; Soares, Rossy-Eric Pereira; Castelo Branco, Patrícia Valéria Gomes; Pereira, Silma Regina Ferreira

    2015-01-01

    Isoflavones are phytoestrogens reported to be potent antioxidant agents. In contrast, the antileishmanial meglumine antimoniate has mutagenic activities. This study evaluated the ability of soy isoflavones to reduce DNA damage induced by meglumine antimoniate. Antimutagenic effects (by micronucleus test) were tested using Swiss mice divided into seven groups treated with meglumine antimoniate (425 mg/kg bw pentavalent antimony); cyclophosphamide (50 mg/kg bw); water (negative control); single isoflavones dose (1.6 mg/kg bw), and three groups received one dose of isoflavones via gavage (0.4 mg/kg bw, 0.8 mg/kg bw or 1.6 mg/kg bw) plus meglumine antimoniate via intraperitoneal, simultaneously. To evaluate antigenotoxicity (by Comet assay), each group with 10 animals received the above-mentioned control doses; single dose of isoflavones 0.8 mg/kg bw, and three groups received isoflavones (0.8 mg/kg bw) by gavage along with intraperitoneal meglumine antimoniate, which were treated with isoflavones 24 h before or after receiving meglumine antimoniate (pre-treatment and post-treatment, respectively) or simultaneously. Cells were harvested 24 h after the treatment, and the data were evaluated by ANOVA followed by Tukey's test (p < 0.05). The data from the simultaneous treatment by micronucleus test revealed that isoflavones (0.4 and 0.8 mg/kg) were able to reverse the mutagenic effect of Glucantime. Moreover, all regimes of the treatment with 0.8 mg/kg bw dose were able to reduce the genotoxicity caused by meglumine antimoniate. It is suggested that the protective effect of isoflavones against DNA damage is related to their ability to reduce oxidative stress caused by the trivalent Sb(III) metabolite of meglumine antimoniate. PMID:25268948

  11. Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk

    PubMed Central

    Aqil, Farrukh; Munagala, Radha; Vadhanam, Manicka V.; Kausar, Hina; Jeyabalan, Jeyaprakash; Schultz, David J.; Gupta, Ramesh C.

    2012-01-01

    Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08 g of light brown powder (purity > 97%) from a total of 40 g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICR-MS analysis. PC (10–40 µM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40 µM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4-hydroxy-17β-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79 µmol of TE/g), as well as by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4 µg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity. PMID:23493479

  12. Comparison of antioxidant enzyme activities and DNA damage in chickpea (Cicer arietinum L.) genotypes exposed to vanadium.

    PubMed

    Imtiaz, Muhammad; Mushtaq, Muhammad Adnan; Rizwan, Muhammad Shahid; Arif, Muhammad Saleem; Yousaf, Balal; Ashraf, Muhammad; Shuanglian, Xiong; Rizwan, Muhammad; Mehmood, Sajid; Tu, Shuxin

    2016-10-01

    The present study was done to elucidate the effects of vanadium (V) on photosynthetic pigments, membrane damage, antioxidant enzymes, protein, and deoxyribonucleic acid (DNA) integrity in the following chickpea genotypes: C-44 (tolerant) and Balkasar (sensitive). Changes in these parameters were strikingly dependent on levels of V, at 60 and 120 mg V L(-1) induced DNA damage in Balkasar only, while photosynthetic pigments and protein were decreased from 15 to 120 mg V L(-1) and membrane was also damaged. It was shown that photosynthetic pigments and protein production declined from 15 to 120 mg V L(-1) and the membrane was also damaged, while DNA damage was not observed at any level of V stress in C-44. Moreover, the antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased in both genotypes of chickpea against V stress; however, more activities were observed in C-44 than Balkasar. The results suggest that DNA damage in sensitive genotypes can be triggered due to exposure of higher vanadium. PMID:27411539

  13. Comparison of antioxidant enzyme activities and DNA damage in chickpea (Cicer arietinum L.) genotypes exposed to vanadium.

    PubMed

    Imtiaz, Muhammad; Mushtaq, Muhammad Adnan; Rizwan, Muhammad Shahid; Arif, Muhammad Saleem; Yousaf, Balal; Ashraf, Muhammad; Shuanglian, Xiong; Rizwan, Muhammad; Mehmood, Sajid; Tu, Shuxin

    2016-10-01

    The present study was done to elucidate the effects of vanadium (V) on photosynthetic pigments, membrane damage, antioxidant enzymes, protein, and deoxyribonucleic acid (DNA) integrity in the following chickpea genotypes: C-44 (tolerant) and Balkasar (sensitive). Changes in these parameters were strikingly dependent on levels of V, at 60 and 120 mg V L(-1) induced DNA damage in Balkasar only, while photosynthetic pigments and protein were decreased from 15 to 120 mg V L(-1) and membrane was also damaged. It was shown that photosynthetic pigments and protein production declined from 15 to 120 mg V L(-1) and the membrane was also damaged, while DNA damage was not observed at any level of V stress in C-44. Moreover, the antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased in both genotypes of chickpea against V stress; however, more activities were observed in C-44 than Balkasar. The results suggest that DNA damage in sensitive genotypes can be triggered due to exposure of higher vanadium.

  14. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    SciTech Connect

    Kiran, Shashi; Oddi, Vineesha; Ramakrishna, Gayatri

    2015-02-01

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  15. SWI/SNF complexes are required for full activation of the DNA-damage response.

    PubMed

    Smith-Roe, Stephanie L; Nakamura, Jun; Holley, Darcy; Chastain, Paul D; Rosson, Gary B; Simpson, Dennis A; Ridpath, John R; Kaufman, David G; Kaufmann, William K; Bultman, Scott J

    2015-01-20

    SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function.

  16. Mutagenicity and DNA-damaging activity of decomposed products of food colours under UV irradiation.

    PubMed

    Ozaki, A; Kitano, M; Itoh, N; Kuroda, K; Furusawa, N; Masuda, T; Yamaguchi, H

    1998-01-01

    Five synthetic food colours Food Red Nos 3, 40 and 102 and Food Blue Nos 1 and 2, and their UV irradiated products were tested for mutagenic activity by means of the Ames test using Salmonella typhimurium strains TA98 and TA100. Food colours were irradiated with UV light for 14 days. Food Red Nos 3, 40 and 102 and Food Blue No. 1 were non-mutagenic before and after irradiation. UV irradiated products of Food Blue No. 2 were mutagenic in TA98 with or without S-9 mix. The mutagenic activity increased with increasing irradiation period, reached maximum potency on day 6, and then decreased. Moreover, Food Blue No. 2 showed DNA-damaging activity after 14 days of irradiation in rec-assay using Bacillus subtilis strains H17 and M45. The capillary electrophoresis was applied for the analysis of UV irradiated products of Food Blue No. 2. The original peak of Food Blue No. 2 was decomposed into seven peaks after UV irradiation.

  17. Regulation of Rad6/Rad18 Activity During DNA Damage Tolerance.

    PubMed

    Hedglin, Mark; Benkovic, Stephen J

    2015-01-01

    Replicative polymerases (pols) cannot accommodate damaged template bases, and these pols stall when such offenses are encountered during S phase. Rather than repairing the damaged base, replication past it may proceed via one of two DNA damage tolerance (DDT) pathways, allowing replicative DNA synthesis to resume. In translesion DNA synthesis (TLS), a specialized TLS pol is recruited to catalyze stable, yet often erroneous, nucleotide incorporation opposite damaged template bases. In template switching, the newly synthesized sister strand is used as a damage-free template to synthesize past the lesion. In eukaryotes, both pathways are regulated by the conjugation of ubiquitin to the PCNA sliding clamp by distinct E2/E3 pairs. Whereas monoubiquitination by Rad6/Rad18 mediates TLS, extension of this ubiquitin to a polyubiquitin chain by Ubc13-Mms2/Rad5 routes DDT to the template switching pathway. In this review, we focus on the monoubiquitination of PCNA by Rad6/Rad18 and summarize the current knowledge of how this process is regulated. PMID:26098514

  18. Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage.

    PubMed

    Acevedo, Julyana; Yan, Shan; Michael, W Matthew

    2016-06-17

    A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.

  19. Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma

    PubMed Central

    Lee, Chang-Lung; Castle, Katherine D.; Moding, Everett J.; Blum, Jordan M.; Williams, Nerissa; Luo, Lixia; Ma, Yan; Borst, Luke B.; Kim, Yongbaek; Kirsch, David G.

    2015-01-01

    Genotoxic cancer therapies, such as chemoradiation, cause haematological toxicity primarily by activating the tumour suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates haematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible p53 short hairpin RNA (shRNA) to show that temporarily blocking p53 during total-body irradiation (TBI) not only ameliorates acute toxicity, but also improves long-term survival by preventing lymphoma development. Using KrasLA1 mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic KrasG12D. However, blocking p53 during TBI significantly suppresses the expansion of KrasG12D-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma. PMID:26399548

  20. Ebselen attenuates oxidative DNA damage and enhances its repair activity in the thalamus after focal cortical infarction in hypertensive rats.

    PubMed

    He, Meixia; Xing, Shihui; Yang, Bo; Zhao, Liqun; Hua, Haiying; Liang, Zhijian; Zhou, Wenliang; Zeng, Jinsheng; Pei, Zhong

    2007-11-21

    Oxidative DNA damage has been proposed to be a major contributor to focal cerebral ischemic injury. However, little is known about the role of oxidative DNA damage in remote damage secondary to the primary infarction. In the present study, we investigated oxidative damage within the ventroposterior nucleus (VPN) after distal middle cerebral artery occlusion (MCAO) in hypertensive rats. We also examined the possible protective effect of ebselen, one glutathione peroxidase mimic, on delayed degeneration in the VPN after distal MCAO. Neuronal damage in the ipsilateral VPN was examined by Nissl staining. Oxidative DNA damage and base repair enzyme activity were assessed by analyzing immunoreactivity of 8-hydroxy-2'-deoxyguanosine (8-ohdG) and 8-oxoguanine DNA glycosylase (OGG1), respectively. The number of intact neurons in the ipsilateral VPN decreased by 52% compared to the contralateral side in ischemia group 2 weeks after distal cerebral cortical infarction. The immunoreactivity of 8-ohdG significantly increased while OGG1 immunoreactivity significantly decreased in the ipsilateral VPN 2 weeks after distal cortical infarction (all p<0.01). Compared with vehicle treatment, ebselen significantly attenuated the neuron loss, ameliorated ischemia-induced increase in 8-ohdG level as well as decrease in OGG1 level within the ipsilateral VPN (all p<0.01). OGG1 was further demonstrated to mainly express in neurons. These findings strongly suggest that oxidative DNA damage may be involved in the delayed neuronal death in the VPN region following distal MCAO. Furthermore, ebselen protects against the delayed damage in the VPN when given at 24 h following distal MCAO.

  1. Aluminum induces oxidative burst, cell wall NADH peroxidase activity, and DNA damage in root cells of Allium cepa L.

    PubMed

    Achary, V Mohan M; Parinandi, Narasimham L; Panda, Brahma B

    2012-08-01

    Plants under stress incur an oxidative burst that involves a rapid and transient overproduction of reactive oxygen species (ROS: O(2) (•-) , H(2) O(2) , (•) OH). We hypothesized that aluminum (Al), an established soil pollutant that causes plant stress, would induce an oxidative burst through the activation of cell wall-NADH peroxidase (NADH-PX) and/or plasma membrane-associated NADPH oxidase (NADPH-OX), leading to DNA damage in the root cells of Allium cepa L. Growing roots of A. cepa were treated with Al(3+) (800 μM of AlCl(3) ) for 3 or 6 hr without or with the pretreatment of inhibitors specific to NADH-PX and NADPH-OX for 2 hr. At the end of the treatment, the extent of ROS generation, cell death, and DNA damage were determined. The cell wall-bound protein (CWP) fractions extracted from the untreated control and the Al-treated roots under the aforementioned experimental conditions were also subjected to in vitro studies, which measured the extent of activation of peroxidase/oxidase, generation of (•) OH, and DNA damage. Overall, the present study demonstrates that the cell wall-bound NADH-PX contributes to the Al-induced oxidative burst through the generation of ROS that lead to cell death and DNA damage in the root cells of A. cepa. Furthermore, the in vitro studies revealed that the CWP fraction by itself caused DNA damage in the presence of NADH, supporting a role for NADH-PX in the stress response. Altogether, this study underscores the crucial function of the cell wall-bound NADH-PX in the oxidative burst-mediated cell death and DNA damage in plants under Al stress.

  2. Telomerase activation as a repair response to radiation-induced DNA damage in Y79 retinoblastoma cells.

    PubMed

    Akiyama, Masaharu; Ozaki, Kohji; Kawano, Takeshi; Yamada, Osamu; Kawauchi, Kiyotaka; Ida, Hiroyuki; Yamada, Hisashi

    2013-10-28

    The molecular mechanism of telomerase activation induced by ionizing radiation (IR) remains poorly understood. We demonstrate that DNA damage induced by IR at doses of 2-5 Gy triggers activation of Akt, predominant to that of protein phosphatase 2A (PP2A), resulting in human telomerase reverse transcriptase (hTERT) phosphorylation and increased telomerase activity in Y79 cells. DNA damage induced by IR at doses greater than 10 Gy might trigger PP2A activation, predominant to that of Akt, resulting in hTERT dephosphorylation and decreased telomerase activity. Our results suggest that differential activation of Akt and PP2A may be responsible for telomerase regulation. PMID:23850566

  3. Evaluation of Antioxidant and DNA Damage Protection Activity of the Hydroalcoholic Extract of Desmostachya bipinnata L. Stapf

    PubMed Central

    Bhimathati, Solomon Sunder Raj

    2014-01-01

    Desmostachya bipinnata Stapf (Poaceae/Gramineae) is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18 ± 3.47 μg/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton's reagent) at a concentration of 50 μg/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea) in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2). Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases. PMID:24574873

  4. 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.

  5. Oxidative DNA Damage Mediated by Intranuclear MMP Activity Is Associated with Neuronal Apoptosis in Ischemic Stroke

    PubMed Central

    Kimura-Ohba, Shihoko

    2016-01-01

    Evidence of the pathological roles of matrix metalloproteinases (MMPs) in various neurological disorders has made them attractive therapeutic targets. MMPs disrupt the blood-brain barrier and cause neuronal death and neuroinflammation in acute cerebral ischemia and are critical for angiogenesis during recovery. However, some challenges have to be overcome before MMPs can be further validated as drug targets in stroke injury. Identifying in vivo substrates of MMPs should greatly improve our understanding of the mechanisms of ischemic injury and is critical for providing more precise drug targets. Recent works have uncovered nontraditional roles for MMPs in the cytosol and nucleus. These have shed light on intracellular targets and biological actions of MMPs, adding additional layers of complexity for therapeutic MMP inhibition. In this review, we discussed the recent advances made in understanding nuclear location of MMPs, their regulation of intranuclear sorting, and their intranuclear proteolytic activity and substrates. In particular, we highlighted the roles of intranuclear MMPs in oxidative DNA damage, neuronal apoptosis, and neuroinflammation at an early stage of stroke insult. These novel data point to new putative MMP-mediated intranuclear actions in stroke-induced pathological processes and may lead to novel approaches to treatment of stroke and other neurological diseases. PMID:26925194

  6. Oxidative DNA damage contributes to the toxic activity of propylparaben in mammalian cells.

    PubMed

    Pérez Martín, José Manuel; Peropadre, Ana; Herrero, Oscar; Fernández Freire, Paloma; Labrador, Verónica; Hazen, María José

    2010-09-30

    Propyl p-hydroxybenzoate, commonly referred to as propylparaben, is the most frequently used preservative to inhibit microbial growth and extend shelf life of a range of consumer products. The objective of this study was to provide further insight into the toxicological profile of this compound, because of the current discrepancy in the literature with regard to the safety of parabens. The Vero cell line, derived from the kidney of the green monkey, was selected to evaluate the adverse effects of propylparaben by use of a set of mechanistically relevant endpoints for detecting cytotoxicity and genotoxic activities. Our results demonstrate that exposure to the compound for 24h causes changes in cell-proliferation rates rather than in cell viability. A significant and dose-dependent decline in the percentage of mitotic cells was observed at the lowest concentration tested, mainly due to cell-cycle arrest at the G0/G1 phase. Immunodetection techniques revealed that induction of DNA double-strand breaks and oxidative damage underlies the cytostatic effect observed in treated Vero cells. Additional studies are in progress to extend these findings, which define a novel mode of action of propylparaben in cultured mammalian cells.

  7. Activation of DNA Damage Response Induced by the Kaposi’s Sarcoma-Associated Herpes Virus

    PubMed Central

    Di Domenico, Enea Gino; Toma, Luigi; Bordignon, Valentina; Trento, Elisabetta; D’Agosto, Giovanna; Cordiali-Fei, Paola; Ensoli, Fabrizio

    2016-01-01

    The human herpes virus 8 (HHV-8), also known as Kaposi sarcoma-associated herpes virus (KSHV), can infect endothelial cells often leading to cell transformation and to the development of tumors, namely Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and the plasmablastic variant of multicentric Castleman’s disease. KSHV is prevalent in areas such as sub-Saharan Africa and the Mediterranean region presenting distinct genotypes, which appear to be associated with differences in disease manifestation, according to geographical areas. In infected cells, KSHV persists in a latent episomal form. However, in a limited number of cells, it undergoes spontaneous lytic reactivation to ensure the production of new virions. During both the latent and the lytic cycle, KSHV is programmed to express genes which selectively modulate the DNA damage response (DDR) through the activation of the ataxia telangiectasia mutated (ATM) pathway and by phosphorylating factors associated with the DDR, including the major tumor suppressor protein p53 tumor suppressor p53. This review will focus on the interplay between the KSHV and the DDR response pathway throughout the viral lifecycle, exploring the putative molecular mechanism/s that may contribute to malignant transformation of host cells. PMID:27258263

  8. MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response.

    PubMed

    Petroni, Marialaura; Veschi, Veronica; Prodosmo, Andrea; Rinaldo, Cinzia; Massimi, Isabella; Carbonari, Maurizio; Dominici, Carlo; McDowell, Heather P; Rinaldi, Christian; Screpanti, Isabella; Frati, Luigi; Bartolazzi, Armando; Gulino, Alberto; Soddu, Silvia; Giannini, Giuseppe

    2011-01-01

    MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma.

  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. Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    PubMed Central

    Jacob, Kimberly D.; Hooten, Nicole Noren; Tadokoro, Takashi; Lohani, Althaf; Barnes, Janice; Evans, Michele K.

    2013-01-01

    Brain tissues from Alzheimer’s Disease (AD) patients show increased levels of oxidative DNA damage and 7,8-dihydro-8-oxoguanine (8-oxoG) accumulation. In humans, the base excision repair protein 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme that recognizes and excises the mutagenic DNA base lesion 8-oxoG. Recently, two polymorphisms of OGG1, A53T and A288V, have been identified in brain tissues of AD patients, but little is known about how these polymorphisms may contribute to AD. We characterized the A53T and A288V polymorphic variants and detected a significant reduction in the catalytic activity for both proteins in vitro and in cells. Additionally, the A53T polymorphism has decreased substrate binding, while the A288V polymorphism has reduced AP lyase activity. Both variants have decreased binding to known OGG1 binding partners PARP-1 and XRCC1. We found that OGG1−/− cells expressing A53T and A288V OGG1 were significantly more sensitive to DNA damage and had significantly decreased survival. Our results provide both biochemical and cellular evidence that A53T and A288V polymorphic proteins have deficiencies in catalytic and protein binding activities that could be related to the increase in oxidative damage to DNA found in AD brains. PMID:23684897

  11. 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

  12. Mechanism of DNA damage tolerance.

    PubMed

    Bi, Xin

    2015-08-26

    DNA damage may compromise genome integrity and lead to cell death. Cells have evolved a variety of processes to respond to DNA damage including damage repair and tolerance mechanisms, as well as damage checkpoints. The DNA damage tolerance (DDT) pathway promotes the bypass of single-stranded DNA lesions encountered by DNA polymerases during DNA replication. This prevents the stalling of DNA replication. Two mechanistically distinct DDT branches have been characterized. One is translesion synthesis (TLS) in which a replicative DNA polymerase is temporarily replaced by a specialized TLS polymerase that has the ability to replicate across DNA lesions. TLS is mechanistically simple and straightforward, but it is intrinsically error-prone. The other is the error-free template switching (TS) mechanism in which the stalled nascent strand switches from the damaged template to the undamaged newly synthesized sister strand for extension past the lesion. Error-free TS is a complex but preferable process for bypassing DNA lesions. However, our current understanding of this pathway is sketchy. An increasing number of factors are being found to participate or regulate this important mechanism, which is the focus of this editorial. PMID:26322163

  13. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    SciTech Connect

    Jarvis, Ian W.H.; Bergvall, Christoffer; Bottai, Matteo; Westerholm, Roger; Stenius, Ulla; Dreij, Kristian

    2013-02-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  14. 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.

  15. Pb-inhibited mitotic activity in onion roots involves DNA damage and disruption of oxidative metabolism.

    PubMed

    Kaur, Gurpreet; Singh, Harminder Pal; Batish, Daizy Rani; Kohli, Ravinder Kumar

    2014-09-01

    Plant responses to abiotic stress significantly affect the development of cells, tissues and organs. However, no studies correlating Pb-induced mitotic inhibition and DNA damage and the alterations in redox homeostasis during root division per se were found in the literature. Therefore, an experiment was conducted to evaluate the impact of Pb on mitotic activity and the associated changes in the oxidative metabolism in onion roots. The cytotoxic effect of Pb on cell division was assessed in the root meristems of Allium cepa (onion). The mitotic index (MI) was calculated and chromosomal abnormalities were sought. Pb-treatment induced a dose-dependent decrease in MI in the onion root tips and caused mitotic abnormalities such as distorted metaphase, fragments, sticky chromosomes, laggards, vagrant chromosomes and bridges. Single Cell Gel Electrophoresis was also performed to evaluate Pb induced genotoxicity. It was accompanied by altered oxidative metabolism in the onion root tips suggesting the interference of Pb with the redox homeostasis during cell division. There was a higher accumulation of malondialdehyde, conjugated dienes and hydrogen peroxide, and a significant increase in the activities of superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases in Pb-treated onion roots, whereas catalases activity exhibited a decreasing pattern upon Pb exposure. The study concludes that Pb-induced cytotoxicity and genotoxicity in the onion roots is mediated through ROS and is also tightly linked to the cell cycle. The exposure to higher concentrations arrested cell cycle leading to cell death, whereas different repair responses are generated at lower concentrations, thereby allowing the cell to complete the cell cycle.

  16. Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue.

    PubMed

    Berra, Carolina Maria; de Oliveira, Carla Santos; Garcia, Camila Carrião Machado; Rocha, Clarissa Ribeiro Reily; Lerner, Letícia Koch; Lima, Leonardo Carmo de Andrade; Baptista, Maurício da Silva; Menck, Carlos Frederico Martins

    2013-08-01

    The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical

  17. Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes.

    PubMed

    Hasegawa, Tatsuya; Nakashima, Masaya; Suzuki, Yoshiharu

    2016-08-26

    Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE2. In addition, inhibition of DNA damage repair by knockdown of XPA, which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. PMID:27343554

  18. Inhibition of histone deacetylase in cancer cells slows down replication forks, activates dormant origins and induces DNA damage

    PubMed Central

    Conti, Chiara; Leo, Elisabetta; Eichler, Gabriel S.; Sordet, Olivier; Martin, Melvenia M.; Fan, Angela; Aladjem, Mirit I.; Pommier, Yves

    2010-01-01

    Protein acetylation is a reversible process regulated by histone deacetylases (HDACs) that is often altered in human cancers. SAHA (suberoylanilide hydroxamic acid) is the first histone deacetylase inhibitor (HDACi) to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and with single-replicon analyses conducted by genome-wide molecular combing after pulse labeling with two thymidine-analogues. Additionally, nascent strand real-time polymerase chain reaction (RT-PCR) in the human β-globin locus was used to assess the effects of SAHA on replication fork origin firing. We found that pharmacological concentrations of SAHA induce replication-mediated DNA damage, on the basis of single-cell and single-DNA molecule analyses. Molecular combing indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the β-globin locus control region. Our findings indicate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells. PMID:20460513

  19. Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes

    PubMed Central

    2013-01-01

    Background Herbaceous plants containing antioxidants can protect against DNA damage. The purpose of this study was to evaluate the antioxidant substances, antioxidant activity, and protection of DNA from oxidative damage in human lymphocytes induced by hydrogen peroxide (H2O2). Our methods used acidic methanol and water extractions from six herbaceous plants, including Bidens alba (BA), Lycium chinense (LC), Mentha arvensis (MA), Plantago asiatica (PA), Houttuynia cordata (HC), and Centella asiatica (CA). Methods Antioxidant compounds such as flavonol and polyphenol were analyzed. Antioxidant activity was determined by the inhibition percentage of conjugated diene formation in a linoleic acid emulsion system and by trolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidative capacities for protecting human lymphocyte DNA from H2O2-induced strand breaks was evaluated by comet assay. Results The studied plants were found to be rich in flavonols, especially myricetin in BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition, polyphenol abounded in BA and CA. The best conjugated diene formation inhibition percentage was found in the acidic methanolic extract of PA. Regarding TEAC, the best antioxidant activity was generated from the acidic methanolic extract of HC. Water and acidic methanolic extracts of MA and HC both had better inhibition percentages of tail DNA% and tail moment as compared to the rest of the tested extracts, and significantly suppressed oxidative damage to lymphocyte DNA. Conclusion Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA, and the leaves of MA and HC extracts may have excellent potential as functional ingredients representing potential sources of natural antioxidants. PMID:24279749

  20. Determination of Free Radical Scavenging, Antioxidative DNA Damage Activities and Phytochemical Components of Active Fractions from Lansium domesticum Corr. Fruit

    PubMed Central

    Klungsupya, Prapaipat; Suthepakul, Nava; Muangman, Thanchanok; Rerk-Am, Ubon; Thongdon-A, Jeerayu

    2015-01-01

    Lansium domesticum Corr. or “long-kong” is one of the most popular fruits in Thailand. Its peel (skin, SK) and seeds (SD) become waste unless recycled or applied for use. This study was undertaken to determine the bioactivity and phytochemical components of L. domesticum (LD) skin and seed extracts. Following various extraction and fractionation procedures, 12 fractions were obtained. All fractions were tested for antioxidant capacity against O2−• and OH•. It was found that the peel of L. domesticum fruits exhibited higher O2−• and OH• scavenging activity than seeds. High potential antioxidant activity was found in two fractions of 50% ethanol extract of peel followed by ethyl acetate (EA) fractionation (LDSK50-EA) and its aqueous phase (LDSK50-H2O). Therefore, these two active fractions were selected for further studies on their antioxidative activity against DNA damage by hydrogen peroxide (H2O2) in human TK6 cells using comet assay. The comet results revealed DNA-protective activity of both LDSK50-EA and LDSK50-H2O fractions when TK6 human lymphoblast cells were pre-treated at 25, 50, 100, and 200 μg/mL for 24 h prior to H2O2 exposure. The phytochemical analysis illustrated the presence of phenolic substances, mainly scopoletin, rutin, and chlorogenic acid, in these two active fractions. This study generates new information on the biological activity of L. domesticum. It will promote and strengthen the utilization of L. domesticum by-products. PMID:26287238

  1. TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response

    PubMed Central

    Rosales, Kimberly Romero; Reid, Michael A.; Yang, Ying; Tran, Thai Q.; Wang, Wen-I; Lowman, Xazmin; Pan, Min; Kong, Mei

    2015-01-01

    Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response. PMID:26717153

  2. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein

    PubMed Central

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-01-01

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. PMID:26227971

  3. DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae

    PubMed Central

    Bierle, Lindsey A.; Reich, Kira L.; Taylor, Braden E.; Blatt, Eliot B.; Middleton, Sydney M.; Burke, Shawnecca D.; Stultz, Laura K.; Hanson, Pamela K.; Partridge, Janet F.; Miller, Mary E.

    2015-01-01

    Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage–including that induced by many anticancer drugs–results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events. PMID:26375390

  4. Identification of Novel Gene Targets and Functions of p21-Activated Kinase 1 during DNA Damage by Gene Expression Profiling

    PubMed Central

    Motwani, Mona; Li, Da-Qiang; Horvath, Anelia; Kumar, Rakesh

    2013-01-01

    P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1

  5. Study of the DNA damage checkpoint using Xenopus egg extracts.

    PubMed

    Willis, Jeremy; DeStephanis, Darla; Patel, Yogin; Gowda, Vrushab; Yan, Shan

    2012-01-01

    On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin. MMS is an alkylating agent that inhibits DNA replication and activates the ATR

  6. 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.

  7. Mammary Stem Cells and Tumor-Initiating Cells Are More Resistant to Apoptosis and Exhibit Increased DNA Repair Activity in Response to DNA Damage

    PubMed Central

    Chang, Chi-Hsuan; Zhang, Mei; Rajapakshe, Kimal; Coarfa, Cristian; Edwards, Dean; Huang, Shixia; Rosen, Jeffrey M.

    2015-01-01

    Summary Adult stem cells and tumor-initiating cells (TICs) often employ different mechanisms of DNA damage response (DDR) as compared to other tissue cell types. However, little is known about how mammary stem cells (MaSCs) and mammary TICs respond to DNA damage. Using the mouse mammary gland and syngeneic p53-null tumors as models, we investigated the molecular and physiological consequences of DNA damage in wild-type MaSCs, p53-null MaSCs, and p53-null TICs. We showed that wild-type MaSCs and basal cells are more resistant to apoptosis and exhibit increased non-homologous end joining (NHEJ) activity. Loss of p53 in mammary epithelium affected both cell-cycle regulation and DNA repair efficiency. In p53-null tumors, we showed that TICs are more resistant to ionizing radiation (IR) due to decreased apoptosis, elevated NHEJ activity, and more-rapid DNA repair. These results have important implications for understanding DDR mechanisms involved in both tumorigenesis and therapy resistance. PMID:26300228

  8. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage.

    PubMed

    Maya, R; Balass, M; Kim, S T; Shkedy, D; Leal, J F; Shifman, O; Moas, M; Buschmann, T; Ronai, Z; Shiloh, Y; Kastan, M B; Katzir, E; Oren, M

    2001-05-01

    The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.

  9. Replicating Damaged DNA in Eukaryotes

    PubMed Central

    Chatterjee, Nimrat; Siede, Wolfram

    2013-01-01

    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. PMID:24296172

  10. 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.

  11. DNA damage in neurodegenerative diseases.

    PubMed

    Coppedè, Fabio; Migliore, Lucia

    2015-06-01

    Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis, which represent three of the most common neurodegenerative pathologies in humans. PMID:26255941

  12. Recruitment and activation of the ATM kinase in the absence of DNA damage sensors

    PubMed Central

    Hartlerode, Andrea J.; Morgan, Mary J.; Wu, Yipin; Buis, Jeffrey; Ferguson, David O.

    2015-01-01

    Two kinases, ATM and DNA-PKcs, control rapid responses to DNA double-strand breaks (DSBs). The paradigm for ATM control is recruitment and activation by the Mre11–Rad50–NBS1 (MRN) sensor complex, whereas DNA-PKcs requires the sensor Ku (Ku70–Ku80). Using Mus musculus cells harboring targeted mutant alleles of Mre11 and/or Ku70, together with pharmacologic kinase inhibition we demonstrate that ATM can in fact be activated by DSBs in the absence of MRN. When MRN is deficient, DNA-PKcs efficiently substitutes for ATM in facilitating local chromatin responses. Strikingly, in the absence of both MRN and Ku, ATM is recruited to chromatin, phosphorylates H2AX, and triggers the G2/M cell cycle checkpoint, but DNA repair functions of MRN are not restored. This implies that a complex interplay between sensors plays a significant role in ATM control, rather than straightforward recruitment and activation by MRN. PMID:26280532

  13. DNA damage and effects on glutathione-S-transferase activity induced by atrazine exposure in zebrafish (Danio rerio).

    PubMed

    Zhu, Lusheng; Dong, Xiaoli; Xie, Hui; Wang, Jun; Wang, Jinhua; Su, Jun; Yu, Changwei

    2011-10-01

    This study was undertaken to investigate the protective effect of atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine) on the activity of glutathione-S-transferase (GST) and DNA damage in males and females of adult zebrafish (Danio rerio). Zebrafish were exposed to control and three treatments (0.01, 0.1, and 1 mg/L) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, for males, the GST activity at lower atrazine concentrations (0.01 and 0.1 mg/L) was markedly higher than that of the controls throughout the duration of the experiment while there was a significant inhibition of the GST activity at 1 mg/L atrazine at days 5 and 20. For females, a significant increase was detected at 0.1 mg/L on the days 5 and 15 and at 0.01 mg/L on day 20. The DNA damage in zebrafish was evaluated using the comet assay; the olive tail moments obtained for hepatopancreas were enhanced after treatment with different concentrations of atrazine on days 5, 10, 15, 20, and 25. The DNA damage increased with increasing atrazine concentrations, indicating that genotoxicity of atrazine and significant differences was found compared to the controls. In conclusion, these findings provide further evidence of the effects of atrazine on aquatic ecosystems.

  14. Protein kinase C{eta} activates NF-{kappa}B in response to camptothecin-induced DNA damage

    SciTech Connect

    Raveh-Amit, Hadas; Hai, Naama; Rotem-Dai, Noa; Shahaf, Galit; Gopas, Jacob; Livneh, Etta

    2011-08-26

    Highlights: {yields} Protein kinase C-eta (PKC{eta}) is an upstream regulator of the NF-{kappa}B signaling pathway. {yields} PKC{eta} activates NF-{kappa}B in non-stressed conditions and in response to DNA damage. {yields} PKC{eta} regulates NF-{kappa}B by activating I{kappa}B kinase (IKK) and inducing I{kappa}B degradation. -- Abstract: The nuclear factor {kappa}B (NF-{kappa}B) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-{kappa}B in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-{kappa}B regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKC{eta}) regulates NF-{kappa}B upstream signaling by activating the I{kappa}B kinase (IKK) and the degradation of I{kappa}B. Furthermore, PKC{eta} enhances the nuclear translocation and transactivation of NF-{kappa}B under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKC{eta} confers protection against DNA damage-induced apoptosis. Our present study suggests that PKC{eta} is involved in NF-{kappa}B signaling leading to drug resistance.

  15. Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells.

    PubMed

    Rosignoli, P; Fabiani, R; De Bartolomeo, A; Spinozzi, F; Agea, E; Pelli, M A; Morozzi, G

    2001-10-01

    Epidemiological studies support the involvement of short-chain fatty acids (SCFA) in colon physiology and the protective role of butyrate on colon carcinogenesis. Among the possible mechanisms by which butyrate may exert its anti-carcinogenicity an antioxidant activity has been recently suggested. We investigated the effects of butyrate and mixtures of SCFA (butyrate, propionate and acetate) on DNA damage induced by H(2)O(2) in isolated human colonocytes and in two human colon tumour cell lines (HT29 and HT29 19A). Human colonocytes were isolated from endoscopically obtained samples and the DNA damage was assessed by the comet assay. H(2)O(2) induced DNA damage in normal colonocytes in a dose-dependent manner which was statistically significant at concentrations over 10 microM. At 15 microM H(2)O(2) DNA damage in HT29 and HT29 19A cells was significantly lower than that observed in normal colonocytes (P < 0.01). Pre-incubation of the cells with physiological concentrations of butyrate (6.25 and 12.5 mM) reduced H(2)O(2) (15 microM) induced damage by 33 and 51% in human colonocytes, 45 and 75% in HT29 and 30 and 80% in HT29 19A, respectively. Treatment of cells with a mixture of 25 mM acetate + 10.4 mM propionate + 6.25 mM butyrate did not induce DNA damage, while a mixture of 50 mM acetate + 20.8 mM propionate + 12.5 mM butyrate was weakly genotoxic only towards normal colonocytes. However, both mixtures were able to reduce the H(2)O(2)-induced DNA damage by about 50% in all cell types. The reported protective effect of butyrate might be important in pathogenetic mechanisms mediated by reactive oxygen species, and aids understanding of the apparent protection toward colorectal cancer exerted by dietary fibres, which enhance the butyrate bioavailability in the colonic mucosa. PMID:11577008

  16. 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.

  17. Evaluation of the Toxicity, AChE Activity and DNA Damage Caused by Imidacloprid on Earthworms, Eisenia fetida.

    PubMed

    Wang, Kai; Qi, Suzhen; Mu, Xiyan; Chai, Tingting; Yang, Yang; Wang, Dandan; Li, Dongzhi; Che, Wunan; Wang, Chengju

    2015-10-01

    Imidacloprid is a well-known pesticide and it is timely to evaluate its toxicity to earthworms (Eisenia fetida). In the present study, the effect of imidacloprid on reproduction, growth, acetylcholinesterase (AChE) and DNA damage in earthworms was assessed using an artificial soil medium. The median lethal concentration (LC50) and the median number of hatched cocoons (EC50) of imidacloprid to earthworms was 3.05 and 0.92 mg/kg respectively, the lowest observed effect concentration of imidacloprid about hatchability, growth, AChE activity and DNA damage was 0.02, 0.5, 0.1 and 0.5 mg/kg, respectively.

  18. Sperm DNA oxidative damage and DNA adducts.

    PubMed

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

    2015-12-01

    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 sperm.

  19. Sperm DNA oxidative damage and DNA adducts.

    PubMed

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

    2015-12-01

    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 sperm

  20. The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

    PubMed

    Ramírez-Guadiana, Fernando H; Barajas-Ornelas, Rocío Del Carmen; Corona-Bautista, Saúl U; Setlow, Peter; Pedraza-Reyes, Mario

    2016-01-01

    The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells' mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation.

  1. The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation

    PubMed Central

    Corona-Bautista, Saúl U.; Setlow, Peter; Pedraza-Reyes, Mario

    2016-01-01

    The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells’ mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation. PMID:26930481

  2. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

    PubMed

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  3. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents

    PubMed Central

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-01-01

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents. PMID:26158513

  4. Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1

    SciTech Connect

    Kobayashi, Junya Tauchi, Hiroshi; Chen, Benjamin; Bruma, Sandeep; Tashiro, Satoshi; Matsuura, Shinya; Tanimoto, Keiji; Chen, David J.; Komatsu, Kenshi

    2009-03-20

    Phosphorylated histone H2AX ({gamma}-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with {gamma}-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether {gamma}-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a {gamma}-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-{gamma}-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, {gamma}-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.

  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. 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...

  8. Protective activity of C-geranylflavonoid analogs from Paulownia tomentosa against DNA damage in 137Cs irradiated AHH-1 cells.

    PubMed

    Moon, Hyung-In; Jeong, Min Ho; Jo, Wol Soon

    2014-09-01

    Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3'-O-methyl-5'-hydroxydiplacone, 3'-O-methyl-5'-O-methyldiplacone and 3'-O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed. PMID:25918796

  9. DNA damage and repair capacity by comet assay in lymphocytes of white-collar active smokers and passive smokers (non- and ex-smokers) at workplace.

    PubMed

    Fracasso, Maria Enrica; Doria, Denise; Franceschetti, Paola; Perbellini, Luigi; Romeo, Luciano

    2006-12-01

    The comet assay has been widely used to quantify DNA damage in isolated lymphocytes from subjects exposed to several environmental or occupational substances, especially for estimation of oxidative damage in the DNA, which is well-known to be induced by tobacco smoke. Passive smoking or environmental tobacco smoke (ETS) has been included among those substances that cause cancer with sufficient evidence in humans. In this study, we analyzed, by the alkaline version of comet assay, the lymphocyte DNA damage of white-collar active smokers and non- and ex-smokers exposed to ETS at the workplace. We investigated basal DNA damage, DNA oxidation by formamidopyrimidine glycosylase (Fpg), the repair capacity H2O2-induced DNA damage by kinetics studies and lymphocyte GSH levels, the major intracellular defense against exogenous oxidative stress imposed by cigarette smoking. Our results indicated high basal DNA damage with clear significant correlations with urinary nicotine and cotinine, number of cigarettes/day, and an inverse significant correlation with GSH cellular content in active smokers. Significant Fpg-sensitive sites were found in smokers (> 85%), considerably high but not significant in passive non- and ex-smokers (> 51% and 37%, respectively). The DNA repair capacity had seriously decreased in non-smokers > smokers > ex-smokers, while the same damage was repaired in a short time in never smokers. PMID:17027201

  10. Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

    PubMed

    Gao, Rui; Liu, Yongping; Silva-Fernandes, Anabela; Fang, Xiang; Paulucci-Holthauzen, Adriana; Chatterjee, Arpita; Zhang, Hang L; Matsuura, Tohru; Choudhary, Sanjeev; Ashizawa, Tetsuo; Koeppen, Arnulf H; Maciel, Patricia; Hazra, Tapas K; Sarkar, Partha S

    2015-01-01

    Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible

  11. Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

    SciTech Connect

    Saquib, Quaiser; Attia, Sabry M.; Siddiqui, Maqsood A.; Aboul-Soud, Mourad A.M.; Al-Khedhairy, Abdulaziz A.; Giesy, John P.; Musarrat, Javed

    2012-02-15

    Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G{sub 2}/M arrest and appearance of a distinctive SubG{sub 1} peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced

  12. The DNA Damage Response Induces Interferon

    PubMed Central

    Brzostek-Racine, Sabrina; Gordon, Chris; Van Scoy, Sarah; Reich, Nancy C.

    2011-01-01

    This study reveals a new complexity in the cellular response to DNA damage: activation of interferon (IFN) signaling. The DNA damage response involves the rapid recruitment of repair enzymes, and the activation of signal transducers that regulate cell cycle checkpoints and cell survival. To understand the link between DNA damage and innate cellular defense that occurs in response to many viral infections, we evaluated the effects of agents such as etoposide that promote double-stranded DNA breaks. Treatment of human cells with etoposide led to the induction of IFN-stimulated genes, and the IFN-α and IFN-λ genes. The nuclear factor-κB (NF-κB), known to be activated in response to DNA damage, was shown to be a key regulator of this IFN gene induction. Expression of an NF-κB subunit, p65/RelA was sufficient for induction of the human IFN-λ1 gene. In addition, NF-κB was required for the induction of the IFN regulatory factors-1 and -7 that are able to stimulate expression of the IFN-α and IFN-λ genes. Cells that lack the NF-κB essential modulator (NEMO), lack the ability to induce the IFN genes following DNA damage. Breaks in DNA are generated during normal physiological processes of replication, transcription, and recombination, as well as by external genotoxic agents or infectious agents. The significant finding of IFN production as a stress response to DNA damage provides a new perspective on the role of IFN signaling. PMID:22013119

  13. 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.

  14. 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

  15. The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells.

    PubMed

    Carballo, Jesús A; Pincheira, Juana; de la Torre, Consuelo

    2006-01-01

    Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.

  16. DNA damage induction of ribonucleotide reductase.

    PubMed

    Elledge, S J; Davis, R W

    1989-11-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. PMID:2513480

  17. Mitochondrial DNA damage and atherosclerosis.

    PubMed

    Yu, Emma P K; Bennett, Martin R

    2014-09-01

    Mitochondria are often regarded as the cellular powerhouses through their ability to generate ATP, the universal fuel for metabolic processes. However, in recent years mitochondria have been recognised as critical regulators of cell death, inflammation, metabolism, and the generation of reactive oxygen species (ROS). Thus, mitochondrial dysfunction directly promotes cell death, inflammation, and oxidative stress and alters metabolism. These are key processes in atherosclerosis and there is now evidence that mitochondrial DNA (mtDNA) damage leads to mitochondrial dysfunction and promotes atherosclerosis directly. In this review we discuss the recent evidence for and mechanisms linking mtDNA defects and atherosclerosis and suggest areas of mitochondrial biology that are potential therapeutic targets.

  18. Amifostine protection against induced DNA damage in gamma-irradiated Escherichia coli cells depend on recN DNA repair gene product activity.

    PubMed

    Almeida, Eliseo; Fuentes, Jorge Luis; Cuetara, Elizabeth; Prieto, Elio; Llagostera, Montserrat

    2010-04-01

    Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against gamma-rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild-type genotypes and in uvr, recF, recB, recB-recC-recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against gamma-radiation-induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double-strand breaks. The results are discussed in relation to amifostine's chemopreventive potential.

  19. Endothelin-1 protects human melanocytes from UV-induced DNA damage by activating JNK and p38 signalling pathways.

    PubMed

    von Koschembahr, Anne M; Swope, Viki B; Starner, Renny J; Abdel-Malek, Zalfa A

    2015-04-01

    Endothelin-1 is a paracrine factor with mitogenic, melanogenic and survival effects on cultured human melanocytes. We report that endothelin-1 signalling reduced the generation and enhanced the repair of ultraviolet radiation (UV)-induced DNA photoproducts, and inhibited apoptosis of human melanocytes, without increasing cAMP levels, melanin content or proliferation. Treatment with endothelin-1 activated the MAP kinases JNK and p38, as evidenced by phosphorylation of their target, activating transcription factor-2 (ATF-2). Endothelin-1 also enhanced the phosphorylation of JNK, p38 and ATF-2 by UV. The effects of endothelin-1 were dependent on increasing intracellular calcium mobilization by endothelin B receptor signalling. Activation of both JNK and p38 was required for reducing DNA photoproducts, but only JNK partially contributed to the survival effect of endothelin-1. ATF-2 activation depended mainly on JNK, yet was not sufficient for the effect of endothelin-1 on UV-induced DNA damage, suggesting the requirement for other JNK and p38 targets for this effect. Our results underscore the significance of endothelin-1 and endothelin B receptor signalling in reducing the genotoxic effects of UV via activating JNK and p38, hence restoring genomic stability of melanocytes.

  20. In-vitro Evaluation of Protective Effects on DNA Damage and Antioxidative Activities of Ilex Spinigera Loes. Extracts

    PubMed Central

    Mohadjerani, Maryam; Vosoghi Roodgar, Mina

    2016-01-01

    Ilex spinigera (Aquifoliaceae) plant is an evergreen tree or shrub with thick glossy dark green leaves and red fruits. This plant has medicinal properties and has been used traditionally in northern Iran for malaria treatment. The aim of this work is to evaluate the antioxidative activities and the inhibitory effect of I. spinigera on the oxidation of DNA. We have found no reports about the popular use of I. spinigera in terms of its chemistry and biology. In this study we report the antioxidant activity of I. spinigera extracts for the first time. Water, ethanol and methanol were used as extraction solvents. Various experimental models including iron (III) reducing power, total antioxidant capacity, DPPH radical scavenging activity, PAB assay and in-vitro inhibition of AAPH-induced oxidation of DNA were used for characterization of antioxidant activity of the extracts. The three extracts showed varying degrees of efficacy in each assay in a dose-dependent manner. The aqueous extract with the highest content of total phenolics, was the most potent antioxidant in all assays except in DPPH assay. The methanol extract with the highest amount of total flavonoids was the potent scavenger of DPPH radical with an IC50 value of 102.22 ± 0.001 μg/mL. Aqueous extract of I. spinigera also showed the protective effect on DNA damage-induced by AAPH. According to our results, I. spinigera leaves extract have the potential for chemoprotective studies. PMID:27610169

  1. In-vitro Evaluation of Protective Effects on DNA Damage and Antioxidative Activities of Ilex Spinigera Loes. Extracts.

    PubMed

    Mohadjerani, Maryam; Vosoghi Roodgar, Mina

    2016-01-01

    Ilex spinigera (Aquifoliaceae) plant is an evergreen tree or shrub with thick glossy dark green leaves and red fruits. This plant has medicinal properties and has been used traditionally in northern Iran for malaria treatment. The aim of this work is to evaluate the antioxidative activities and the inhibitory effect of I. spinigera on the oxidation of DNA. We have found no reports about the popular use of I. spinigera in terms of its chemistry and biology. In this study we report the antioxidant activity of I. spinigera extracts for the first time. Water, ethanol and methanol were used as extraction solvents. Various experimental models including iron (III) reducing power, total antioxidant capacity, DPPH radical scavenging activity, PAB assay and in-vitro inhibition of AAPH-induced oxidation of DNA were used for characterization of antioxidant activity of the extracts. The three extracts showed varying degrees of efficacy in each assay in a dose-dependent manner. The aqueous extract with the highest content of total phenolics, was the most potent antioxidant in all assays except in DPPH assay. The methanol extract with the highest amount of total flavonoids was the potent scavenger of DPPH radical with an IC50 value of 102.22 ± 0.001 μg/mL. Aqueous extract of I. spinigera also showed the protective effect on DNA damage-induced by AAPH. According to our results, I. spinigera leaves extract have the potential for chemoprotective studies. PMID:27610169

  2. In-vitro Evaluation of Protective Effects on DNA Damage and Antioxidative Activities of Ilex Spinigera Loes. Extracts

    PubMed Central

    Mohadjerani, Maryam; Vosoghi Roodgar, Mina

    2016-01-01

    Ilex spinigera (Aquifoliaceae) plant is an evergreen tree or shrub with thick glossy dark green leaves and red fruits. This plant has medicinal properties and has been used traditionally in northern Iran for malaria treatment. The aim of this work is to evaluate the antioxidative activities and the inhibitory effect of I. spinigera on the oxidation of DNA. We have found no reports about the popular use of I. spinigera in terms of its chemistry and biology. In this study we report the antioxidant activity of I. spinigera extracts for the first time. Water, ethanol and methanol were used as extraction solvents. Various experimental models including iron (III) reducing power, total antioxidant capacity, DPPH radical scavenging activity, PAB assay and in-vitro inhibition of AAPH-induced oxidation of DNA were used for characterization of antioxidant activity of the extracts. The three extracts showed varying degrees of efficacy in each assay in a dose-dependent manner. The aqueous extract with the highest content of total phenolics, was the most potent antioxidant in all assays except in DPPH assay. The methanol extract with the highest amount of total flavonoids was the potent scavenger of DPPH radical with an IC50 value of 102.22 ± 0.001 μg/mL. Aqueous extract of I. spinigera also showed the protective effect on DNA damage-induced by AAPH. According to our results, I. spinigera leaves extract have the potential for chemoprotective studies.

  3. 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.

  4. 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. PMID:27555866

  5. 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

  6. DNA damage response in adult stem cells.

    PubMed

    Insinga, Alessandra; Cicalese, Angelo; Pelicci, Pier Giuseppe

    2014-04-01

    This review discusses the processes of DNA-damage-response and DNA-damage repair in stem and progenitor cells of several tissues. The long life-span of stem cells suggests that they may respond differently to DNA damage than their downstream progeny and, indeed, studies have begun to elucidate the unique stem cell response mechanisms to DNA damage. Because the DNA damage responses in stem cells and progenitor cells are distinctly different, stem and progenitor cells should be considered as two different entities from this point of view. Hematopoietic and mammary stem cells display a unique DNA-damage response, which involves active inhibition of apoptosis, entry into the cell-cycle, symmetric division, partial DNA repair and maintenance of self-renewal. Each of these biological events depends on the up-regulation of the cell-cycle inhibitor p21. Moreover, inhibition of apoptosis and symmetric stem cell division are the consequence of the down-regulation of the tumor suppressor p53, as a direct result of p21 up-regulation. A deeper understanding of these processes is required before these findings can be translated into human anti-aging and anti-cancer therapies. One needs to clarify and dissect the pathways that control p21 regulation in normal and cancer stem cells and define (a) how p21 blocks p53 functions in stem cells and (b) how p21 promotes DNA repair in stem cells. Is this effect dependent on p21s ability to inhibit p53? Such molecular knowledge may pave the way to methods for maintaining short-term tissue reconstitution while retaining long-term cellular and genomic integrity.

  7. 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.

  8. The effects of dietary boric acid and borax supplementation on lipid peroxidation, antioxidant activity, and DNA damage in rats.

    PubMed

    Ince, Sinan; Kucukkurt, Ismail; Cigerci, Ibrahim Hakki; Fatih Fidan, A; Eryavuz, Abdullah

    2010-07-01

    The aims of this study were to clarify the effects of high dietary supplementation with boric acid and borax, called boron (B) compounds, on lipid peroxidation (LPO), antioxidant activity, some vitamin levels, and DNA damage in rats. Thirty Sprague Dawley male rats were divided into three equal groups: the animals in the first group (control) were fed with a standard rodent diet containing 6.4 mg B/kg, and the animals in the experimental group were fed with a standard rodent diet added with a supra-nutritional amount of boric acid and borax (100 mg B/kg) throughout the experimental period of 28 days. The B compounds decreased malondialdehyde (MDA), DNA damage, the protein carbonyl content (PCO) level in blood, and glutathione (GSH) concentration in the liver, Cu-Zn superoxide dismutase (SOD), and catalase (CAT) activity in the kidney. The B compounds increased GSH concentration in blood and the vitamin C level in plasma. Consequently, our results demonstrate that B supplementation (100 mg/kg) in diet decreases LPO, and enhances the antioxidant defense mechanism and vitamin status. There are no differences in oxidant/antioxidant balance and biochemical parameters except for serum vitamin A and liver GSH concentration, between the boron compounds used in this study.

  9. alpha-MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV-induced DNA damage in human melanocytes.

    PubMed

    Abdel-Malek, Zalfa A; Ruwe, Andrew; Kavanagh-Starner, Renny; Kadekaro, Ana Luisa; Swope, Viki; Haskell-Luevano, Carrie; Koikov, Leonid; Knittel, James J

    2009-10-01

    One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of alpha-melanocortin (alpha-MSH) that were more potent and stable than the physiological alpha-MSH, and mimicked its photoprotective effects against UV-induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified alpha-MSH core His(6)-d-Phe(7)-Arg(8), which contained different N-capping groups, C-terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C-terminal modifications. The most effective C-terminal tripeptide mimicked alpha-MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non-functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention. PMID:19558415

  10. alpha-MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV-induced DNA damage in human melanocytes.

    PubMed

    Abdel-Malek, Zalfa A; Ruwe, Andrew; Kavanagh-Starner, Renny; Kadekaro, Ana Luisa; Swope, Viki; Haskell-Luevano, Carrie; Koikov, Leonid; Knittel, James J

    2009-10-01

    One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of alpha-melanocortin (alpha-MSH) that were more potent and stable than the physiological alpha-MSH, and mimicked its photoprotective effects against UV-induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified alpha-MSH core His(6)-d-Phe(7)-Arg(8), which contained different N-capping groups, C-terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C-terminal modifications. The most effective C-terminal tripeptide mimicked alpha-MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non-functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.

  11. Potential effects of environmental contaminants on P450 aromatase activity and DNA damage in swallows from the Rio Grande and Somerville, Texas

    USGS Publications Warehouse

    Sitzlar, M.A.; Mora, M.A.; Fleming, J.G.W.; Bazer, F.W.; Bickham, J.W.; Matson, C.W.

    2009-01-01

    Cliff swallows (Petrochelidon pyrrhonota) and cave swallows (P. fulva) were sampled during the breeding season at several locations in the Rio Grande, Texas, to evaluate the potential effects of environmental contaminants on P450 aromatase activity in brain and gonads and DNA damage in blood cells. The tritiated water-release aromatase assay was used to measure aromatase activity and flow cytometry was used to measure DNA damage in nucleated blood cells. There were no significant differences in brain and gonadal aromatase activities or in estimates of DNA damage (HPCV values) among cave swallow colonies from the Lower Rio Grande Valley (LRGV) and Somerville. However, both brain and gonadal aromatase activities were significantly higher (P < 0.05) in male cliff swallows from Laredo than in those from Somerville. Also, DNA damage estimates were significantly higher (P < 0.05) in cliff swallows (males and females combined) from Laredo than in those from Somerville. Contaminants of current high use in the LRGV, such as atrazine, and some of the highly persistent organochlorines, such as toxaphene and DDE, could be potentially associated with modulation of aromatase activity in avian tissues. Previous studies have indicated possible DNA damage in cliff swallows. We did not observe any differences in aromatase activity or DNA damage in cave swallows that could be associated with contaminant exposure. Also, the differences in aromatase activity and DNA damage between male cliff swallows from Laredo and Somerville could not be explained by contaminants measured at each site in previous studies. Our study provides baseline information on brain and gonadal aromatase activity in swallows that could be useful in future studies. ?? 2008 Springer Science+Business Media, LLC.

  12. Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

    PubMed Central

    Friedman, Joshua I.; Stivers, James T.

    2010-01-01

    A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

  13. Expression of PCNA-binding domain of CtIP, a motif required for CtIP localization at DNA replication foci, causes DNA damage and activation of DNA damage checkpoint.

    PubMed

    Gu, Bingnan; Chen, Phang-Lang

    2009-05-01

    CtIP, CtBP-interacting protein, is a nuclear protein that was identified as a cofactor for the transcriptional repressor CtBP. Our genetic studies in mice revealed that haploid insufficiency of CtIP leads to tumorigenesis and is associated with shortened life span. At the molecular level, CtIP is a multivalent adaptor. It interacts directly with pRB family members, the prototype tumor suppressor proteins, and contributes to G(1)/S regulation. It has also been implicated in DNA damage checkpoint control through its interaction with the breast cancer susceptibility gene product BRCA1. Recently, it was found to modulate the nuclease activity of the Mre11/Rad50/NBS1 complex. Here we report that CtIP is recruited to S-phase DNA replication foci through a novel motif functioning as replication foci targeting sequence (RFTS). This motif contains a consensus PCNA-interacting protein box that binds to PCNA both in vivo and in vitro. In support of the biological significance of this interaction, we detected arrest of the cell cycle at the S/G(2) phase transition, and suppression of cell proliferation in U2-OS cells upon the conditional expression of the wild type, but not a mutated RFTS using a tetracycline-inducible system. We found that cells expressing RFTS had excess DNA double strand breaks as demonstrated by formation of gamma-H2AX nuclear foci. Finally, G(2)/M checkpoint activation in response to the expression of the CtIP RFTS is abrogated by caffeine treatment. Our work suggests an intimate relationship between CtIP and PCNA may be important for the maintenance of genomic stability in higher eukaryotic organism.

  14. DNA damage by bromate: mechanism and consequences.

    PubMed

    Ballmaier, Daniel; Epe, Bernd

    2006-04-17

    Exposure of mammalian cells to bromate (BrO3-) generates oxidative DNA modifications, in particular 7,8-dihydro-8-oxo-guanine (8-oxoG). The damaging mechanism is quite unique, since glutathione, which is protective against most oxidants and alkylating agents, mediates a metabolic activation, while bromate itself does not react directly with DNA. Neither enzymes nor transition metals are required as catalysts in the activation. The ultimate DNA damaging species has not yet been established, but experiments under cell-free conditions suggest that neither molecular bromine nor reactive oxygen species such as superoxide, hydrogen peroxide or singlet oxygen are involved. Rather bromine radicals (Br*) or oxides (BrO*, BrO2*) might be responsible. Compared to hypochlorite (ClO-), bromate is much less cytotoxic, probably because the former halite efficiently reacts with proteins and other vitally important cellular constituents. In consequence, oxidative DNA damage and the induction of mutations and micronuclei is easily detectable at non-cytotoxic concentrations of bromate, while DNA damage by hypochlorite is observed only at cytotoxic concentrations and follows a non-linear (hockey-stick-like) dose response. PMID:16490296

  15. Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells.

    PubMed

    Arlt, Volker M; Cole, Kathleen J; Phillips, David H

    2004-03-01

    3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5. DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis. DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1-50 microM, 2 h incubation time). At 50 microM median comet tail lengths (CTLs) were 25.0 microm for 3-NBA, 48.0 microm for 3-ABA, 54.5 microm for 3-Ac-ABA and 65.0 microm for N-Ac-N-OH-ABA. Median CTLs in control incubations were in the range 7.7-13.1 micro m. Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea. Depending on the concentration used (1-20 microM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay. 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 microM the total number of micronuclei per 500 binucleate cells was 4.7 +/- 0.6 in both cases, compared with 1.7-3.0 for controls (P < 0.05). Furthermore, multiple DNA adducts were detected with each compound (1 microM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites. DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/10(8) nt using the nuclease P1 and butanol enrichment procedures, respectively. DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA

  16. alpha-Melanocortin and endothelin-1 activate antiapoptotic pathways and reduce DNA damage in human melanocytes.

    PubMed

    Kadekaro, Ana Luisa; Kavanagh, Renny; Kanto, Hiromi; Terzieva, Silva; Hauser, Jennifer; Kobayashi, Nobuhiko; Schwemberger, Sandy; Cornelius, James; Babcock, George; Shertzer, Howard G; Scott, Glynis; Abdel-Malek, Zalfa A

    2005-05-15

    UV radiation is an important etiologic factor for skin cancer, including melanoma. Constitutive pigmentation and the ability to tan are considered the main photoprotective mechanism against sun-induced carcinogenesis. Pigmentation in the skin is conferred by epidermal melanocytes that synthesize and transfer melanin to keratinocytes. Therefore, insuring the survival and genomic stability of epidermal melanocytes is critical for inhibiting photocarcinogenesis, particularly melanoma, the most deadly form of skin cancer. The paracrine factors alpha-melanocortin and endothelin-1 are critical for the melanogenic response of cultured human melanocytes to UV radiation. We report that alpha-melanocortin and endothelin-1 rescued human melanocytes from UV radiation-induced apoptosis and reduced DNA photoproducts and oxidative stress. The survival effects of alpha-melanocortin and endothelin-1 were mediated by activation of the melanocortin 1 and endothelin receptors, respectively. Treatment of melanocytes with alpha-melanocortin and/or endothelin-1 before exposure to UV radiation activated the inositol triphosphate kinase-Akt pathway and increased the phosphorylation and expression of the microphthalmia-related transcription factor. Treatment with alpha-melanocortin and/or endothelin-1 enhanced the repair of cyclobutane pyrimidine dimers and reduced the levels of hydrogen peroxide induced by UV radiation. These effects are expected to reduce genomic instability and mutagenesis.

  17. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    PubMed

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  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. PMID:25434743

  19. Embryonic stem cells lacking the epigenetic regulator Cfp1 are hypersensitive to DNA-damaging agents and exhibit decreased Ape1/Ref-1 protein expression and endonuclease activity.

    PubMed

    Tate, Courtney M; Fishel, Melissa L; Holleran, Julianne L; Egorin, Merrill J; Skalnik, David G

    2009-12-01

    Modulation of chromatin structure plays an important role in the recruitment and function of DNA repair proteins. CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is essential for mammalian development and is an important regulator of chromatin structure. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1(-/-)) are viable but demonstrate a dramatic decrease in cytosine methylation, altered histone methylation, and an inability to differentiate. We find that ES cells lacking Cfp1 are hypersensitive to a variety of DNA-damaging agents. In addition, CXXC1(-/-) ES cells accumulate more DNA damage and exhibit decreased protein expression and endonuclease activity of AP endonuclease (Ape1/Ref-1), an enzyme involved in DNA base excision repair. Expression in CXXC1(-/-) ES cells of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) restores normal Ape1/Ref-1 protein expression and rescues the hypersensitivity to DNA-damaging agents, demonstrating that Cfp1 contains redundant functional domains. Furthermore, retention of either the DNA-binding activity of Cfp1 or interaction with the Setd1A and Setd1B histone H3-Lys4 methyltransferase complexes is required to restore normal sensitivity of CXXC1(-/-) ES cells to DNA-damaging agents. These results implicate Cfp1 as a regulator of DNA repair processes. PMID:19836314

  20. 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.

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

    SciTech Connect

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-07-01

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

  2. Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

    PubMed Central

    2016-01-01

    ABSTRACT There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized. PMID:27624129

  3. Extracellular ATP protects endothelial cells against DNA damage.

    PubMed

    Aho, Joonas; Helenius, Mikko; Vattulainen-Collanus, Sanna; Alastalo, Tero-Pekka; Koskenvuo, Juha

    2016-09-01

    Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers γH2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.

  4. Yap1: A DNA damage responder in Saccharomyces cerevisiae

    PubMed Central

    Rowe, Lori A.; Degtyareva, Natalya; Doetsch, Paul W.

    2012-01-01

    Activation of signaling pathways in response to genotoxic stress is crucial for cells to properly repair DNA damage. In response to DNA damage, intracellular levels of reactive oxygen species increase. One important function of such a response could be to initiate signal transduction processes. We have employed the model eukaryote Saccharomyces cerevisiae to delineate DNA damage sensing mechanisms. We report a novel, unanticipated role for the transcription factor Yap1 as a DNA damage responder, providing direct evidence that reactive oxygen species are an important component of the DNA damage signaling process. Our findings reveal an epistatic link between Yap1 and the DNA base excision repair pathway. Corruption of the Yap1-mediated DNA damage response influences cell survival and genomic stability in response to exposure to genotoxic agents. PMID:22433435

  5. Yap1: a DNA damage responder in Saccharomyces cerevisiae.

    PubMed

    Rowe, Lori A; Degtyareva, Natalya; Doetsch, Paul W

    2012-04-01

    Activation of signaling pathways in response to genotoxic stress is crucial for cells to properly repair DNA damage. In response to DNA damage, intracellular levels of reactive oxygen species increase. One important function of such a response could be to initiate signal transduction processes. We have employed the model eukaryote Saccharomyces cerevisiae to delineate DNA damage sensing mechanisms. We report a novel, unanticipated role for the transcription factor Yap1 as a DNA damage responder, providing direct evidence that reactive oxygen species are an important component of the DNA damage signaling process. Our findings reveal an epistatic link between Yap1 and the DNA base excision repair pathway. Corruption of the Yap1-mediated DNA damage response influences cell survival and genomic stability in response to exposure to genotoxic agents.

  6. Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

    PubMed Central

    Patel, Yogin; Gowda, Vrushab; Yan, Shan

    2012-01-01

    On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates

  7. 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

  8. 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

  9. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    PubMed Central

    Ruiz-Magaña, María J.; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-01-01

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect. PMID:26942461

  10. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells.

    PubMed

    Ruiz-Magaña, María J; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-04-19

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect.

  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. PMID:26046974

  12. DNA repair of oxidative DNA damage in human carcinogenesis

    PubMed Central

    Paz-Elizur, Tamar; Sevilya, Ziv; Leitner-Dagan, Yael; Elinger, Dalia; Roisman, Laila; Livneh, Zvi

    2008-01-01

    Efficient DNA repair mechanisms comprise a critical component in the protection against human cancer, as indicated by the high predisposition to cancer of individuals with germ-line mutations in DNA repair genes. This includes biallelic germ-line mutations in the MUYH gene, encoding a DNA glycosylase that is involved in the repair of oxidative DNA damage, which strongly predispose humans to a rare hereditary form of colorectal cancer. Extensive research efforts including biochemical, enzymological and genetic studies in model organisms established that the oxidative DNA lesion 8-oxoguanine is mutagenic, and that several DNA repair mechanisms operate to prevent its potentially mutagenic and carcinogenic outcome. Epidemiological studies on the association with sporadic cancers of single nucleotide polymorphisms in genes such as OGG1, involved in the repair of 8-oxoguanine yielded conflicting results, and suggest a minor effect at best. A new approach based on the functional analysis of DNA repair enzymatic activity showed that reduced activity of 8-oxoguanine DNA glycosylase (OGG) is a risk factor in lung and head and neck cancer. Moreover, the combination of smoking and low OGG activity was associated with a higher risk, suggesting a potential strategy for risk assessment and prevention of lung cancer, as well as other types of cancer. PMID:18374480

  13. 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. PMID:27236213

  14. Effects of ultraviolet A on the activity of two metabolic enzymes, DNA damage and lipid peroxidation during early developmental stages of the African catfish, Clarias gariepinus (Burchell, 1822)

    PubMed Central

    Mekkawy, Imam A. A.; Mahmoud, Usama M.; Osman, Alaa G.

    2009-01-01

    Many ultraviolet-A (UVA)-induced biochemical and physiological changes are valid as biomarkers using aquatic species for detection of the degree of stress. Changes in the concentration and activities of enzymes, such as glucose-6-phosphate dehyderogenase (G6PDH), lactate dehyderogenase (LDH), DNA damage and lipid peroxidation (LPO), can be used as biomarkers to identify possible environmental contamination in fish. This study aimed to investigate the impact of UVA on the activity of the selected enzymes, DNA damage and LPO during early developmental stages of the African catfish Clarias gariepinus. Embryo hemogenates were used for measurements of G6PDH, LDH, DNA damage and LPO concentrations and activities spectrophotometrically at 37°C. The normal ontogenetic variations in enzyme activities, DNA damage and LPO of the early developmental stages (24–168 h-PFS; hours-post fertilization stage) were studied. There was a significant decrease in the activity of G6PDH till 120 h-PFS. Then after 120 h-PFS, the activity of such enzymes insignificantly increased toward higher stages. The LDH activity was recorded with a pattern of decrease till 96 h-PFS, followed by a significant increase toward 168 h-PFS. The polynomial pattern of variations in DNA damage and LPO was also evident. The patterns of the enzyme activities, corresponding DNA damage and LPO of the early ontogenetic stages under the influence of three different UVA doses (15, 30 and 60 min), were recorded. The pattern of variations in G6PDH activity in UVA-induced groups was similar to that of the control group with variation in the magnitude of such activity. In all treated groups, LDH activity decreased till 96 h-PFS, then increased till 168 h-PFS. Within each of the embryonic stages, the increase in UVA led to a significant increase in DNA damage. A significant increase in lipid peroxidation under UVA doses was recorded. The variability in number and molecular weight of proteins under exposure to UVA

  15. WISP-1 attenuates p53-mediated apoptosis in response to DNA damage through activation of the Akt kinase.

    PubMed

    Su, Fei; Overholtzer, Michael; Besser, Daniel; Levine, Arnold J

    2002-01-01

    WISP-1 (Wnt-1-induced secreted protein) was identified as an oncogene regulated by the Wnt-1-beta-catenin pathway. WISP-1 belongs to the CCN family of growth factors, which are cysteine-rich, heparin-binding, secreted proteins associated with the extracellular matrix, and can interact with cellular integrins. Expression of WISP-1 in some cells results in transformation and tumorigenesis. Here it is shown that WISP-1 can activate the antiapoptotic Akt/PKB signaling pathway. It also is demonstrated that WISP-1 can prevent cells from undergoing apoptosis following DNA damage through inhibition of the mitochondrial release of cytochrome c and up-regulation of antiapoptotic Bcl-X(L). Furthermore, the results show that WISP-1 protects cells from p53-dependent cell death, but not Fas-ligand activated cell death, suggesting that there may be cross talk between the tumor suppressor protein p53 and WISP-1 signaling pathways. WISP-1 acts to block cell death at a late stage in the p53-mediated apoptosis pathway.

  16. Telomerase Reverse Transcriptase and Peroxisome Proliferator-Activated Receptor γ Co-Activator-1α Cooperate to Protect Cells from DNA Damage and Mitochondrial Dysfunction in Vascular Senescence.

    PubMed

    Mendelsohn, Andrew R; Larrick, James W

    2015-10-01

    Reduced telomere length with increasing age in dividing cells has been implicated in contributing to the pathologies of human aging, which include cardiovascular and metabolic disorders, through induction of cellular senescence. Telomere shortening results from the absence of telomerase, an enzyme required to maintain telomere length. Telomerase reverse transcriptase (TERT), the protein subunit of telomerase, is expressed only transiently in a subset of adult somatic cells, which include stem cells and smooth muscle cells. A recent report from Xiong and colleagues demonstrates a pivotal role for the transcription co-factor peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) in maintaining TERT expression and preventing vascular senescence and atherosclerosis in mice. Ablation of PGC-1α reduced TERT expression and increased DNA damage and reactive oxygen species (ROS), resulting in shortened telomeres and vascular senescence. In the ApoE(-/-) mouse model of atherosclerosis, forced expression of PGC-1α increased expression of TERT, extended telomeres, and reversed genomic DNA damage, vascular senescence, and the development of atherosclerotic plaques. Alpha lipoic acid (ALA) stimulated expression of PGC-1α and TERT and reversed DNA damage, vascular senescence, and atherosclerosis, similarly to ectopic expression of PGC-1α. ALA stimulated cyclic adenosine monophosphate (cAMP) signaling, which in turn activated the cAMP response element-binding protein (CREB), a co-factor for PGC-1α expression. The possibility that ALA might induce TERT to extend telomeres in human cells suggests that ALA may be useful in treating atherosclerosis and other aging-related diseases. However, further investigation is needed to identify whether ALA induces TERT in human cells, which cell types are susceptible, and whether such changes have clinical significance. PMID:26414604

  17. 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

  18. Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints.

    PubMed

    Wang, Hui-Min; Cheng, Kuo-Chen; Lin, Cheng-Jung; Hsu, Shu-Wei; Fang, Wei-Cheng; Hsu, Tai-Feng; Chiu, Chien-Chih; Chang, Hsueh-Wei; Hsu, Chun-Hua; Lee, Alan Yueh-Luen

    2010-12-01

    Several compounds from Cinnamomum kotoense show anticancer activities. However, the detailed mechanisms of most compounds from C. kotoense remain unknown. In this study, we investigated the anticancer activity of obtusilactone A (OA) and (-)-sesamin in lung cancer. Our results show that human Lon is upregulated in non-small-cell lung cancer (NSCLC) cell lines, and downregulation of Lon triggers caspase-3 mediated apoptosis. Through enzyme-based screening, we identified two small-molecule compounds, obtusilactone A (OA) and (-)-sesamin from C. kotoense, as potent Lon protease inhibitors. Obtusilactone A and (-)-sesamin interact with Ser855 and Lys898 residues in the active site of the Lon protease according to molecular docking analysis. Thus, we suggest that cancer cytotoxicity of the compounds is partly due to the inhibitory effects on Lon protease. In addition, the compounds are able to cause DNA double-strand breaks and activate checkpoints. Treatment with OA and (-)-sesamin induced p53-independent DNA damage responses in NSCLC cells, including G(1) /S checkpoint activation and apoptosis, as evidenced by phosphorylation of checkpoint proteins (H2AX, Nbs1, and Chk2), caspase-3 cleavage, and sub-G(1) accumulation. In conclusion, OA and (-)-sesamin act as both inhibitors of human mitochondrial Lon protease and DNA damage agents to activate the DNA damage checkpoints as well induce apoptosis in NSCLC cells. These dual functions open a bright avenue to develop more selective chemotherapy agents to overcome chemoresistance and sensitize cancer cells to other chemotherapeutics.

  19. 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

  20. 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.

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

    PubMed Central

    Bakkenist, Christopher J.; Kastan, Michael B.

    2016-01-01

    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

  2. DNA damage-induced replication arrest in Xenopus egg extracts

    PubMed Central

    Stokes, Matthew P.; Michael, W. Matthew

    2003-01-01

    Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase α, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase α and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA. PMID:14581453

  3. Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

    PubMed Central

    Li, Na; Parrish, Marcus; Chan, Tze Khee; Yin, Lu; Rai, Prashant; Yoshiyuki, Yamada; Abolhassani, Nona; Tan, Kong Bing; Kiraly, Orsolya; Chow, Vincent TK; Engelward, Bevin P.

    2016-01-01

    Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe Influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of two weeks in vivo. We show that influenza induces DNA damage both when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage, persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67 positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DSB repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome. PMID:25809161

  4. 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.

  5. 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.

  6. γ-H2AX is a sensitive marker of DNA damage induced by metabolically activated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

    PubMed

    Ibuki, Yuko; Shikata, Mariko; Toyooka, Tatsushi

    2015-10-01

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived nitrosamine, is a potent pulmonary carcinogen present in tobacco smoke. DNA adducts induced by metabolically activated NNK cause carcinogenesis; however, the DNA adducts are difficult to detect in cultured cells because of low intrinsic metabolic enzyme activity. In this study, we indirectly detected NNK-induced DNA adducts via the phosphorylation of histone H2AX (γ-H2AX) in A549 human lung adenocarcinoma epithelial cells. NNK treatment dose-dependently induced γ-H2AX. This γ-H2AX induction was suppressed by ataxia telangiectasia mutated inhibitors, suggesting that DNA double-strand breaks (DSBs) are formed during replication and repair of DNA adducts; however, DSBs could not be directly detected by biased sinusoidal field gel electrophoresis (BSFGE). CYP2A13-overexpressing cells showed prolonged γ-H2AX induction compared with control cells, and DSBs could be detected by BSFGE in CYP2A13-overexpressing cells as a clear migration of double-stranded DNA. These findings suggest that γ-H2AX is a sensitive marker of DNA adducts and provides a possible system for genotoxicity screening of chemicals such as NNK, which need metabolic activation to induce DNA damage. PMID:26231820

  7. At a crossroads: human DNA tumor viruses and the host DNA damage response.

    PubMed

    Nikitin, Pavel A; Luftig, Micah A

    2011-07-01

    Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection. PMID:21927617

  8. Preventing metal-mediated oxidative DNA damage with selenium compounds.

    PubMed

    Battin, Erin E; Zimmerman, Matthew T; Ramoutar, Ria R; Quarles, Carolyn E; Brumaghim, Julia L

    2011-05-01

    Copper and iron are two widely studied transition metals associated with hydroxyl radical (˙OH) generation, oxidative damage, and disease development. Because antioxidants ameliorate metal-mediated DNA damage, DNA gel electrophoresis assays were used to quantify the ability of ten selenium-containing compounds to inhibit metal-mediated DNA damage by hydroxyl radical. In the Cu(I)/H(2)O(2) system, selenocystine, selenomethionine, and methyl-selenocysteine inhibit DNA damage with IC(50) values ranging from 3.34 to 25.1 μM. Four selenium compounds also prevent DNA damage from Fe(II) and H(2)O(2). Additional gel electrophoresis experiments indicate that Cu(I) or Fe(II) coordination is responsible for the selenium antioxidant activity. Mass spectrometry studies show that a 1 : 1 stoichiometry is the most common for iron and copper complexes of the tested compounds, even if no antioxidant activity is observed, suggesting that metal coordination is necessary but not sufficient for selenium antioxidant activity. A majority of the selenium compounds are electroactive, regardless of antioxidant activity, and the glutathione peroxidase activities of the selenium compounds show no correlation to DNA damage inhibition. Thus, metal binding is a primary mechanism of selenium antioxidant activity, and both the chemical functionality of the selenium compound and the metal ion generating damaging hydroxyl radical significantly affect selenium antioxidant behavior. PMID:21286651

  9. Protease Activity of PprI Facilitates DNA Damage Response: Mn(2+)-Dependence and Substrate Sequence-Specificity of the Proteolytic Reaction

    PubMed Central

    Lu, Huiming; Lin, Lin; Wang, Liangyan; Xu, Hong; Cui, Xianyan; Zhang, Hui; Li, Tingting; Hua, Yuejin

    2015-01-01

    The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism. PMID:25811789

  10. Neuroprotective activity of Viola mandshurica extracts on hydrogen peroxide-induced DNA damage and cell death in PC12 cells.

    PubMed

    Jeon, Gyeong-Im; Yoon, Mi-Young; Park, Hae-Ryoung; Lee, Seung-Cheol; Park, Eunju

    2009-08-01

    This study was conducted to examine the neuroprotective effects of acetone extracts from Viola mandshurica (VME). The effect of VME on hydrogen peroxide (H(2)O(2))-induced DNA damage in PC12 cells was evaluated by the comet assay where VME (100 and 250 microg/mL) was a dose-dependent inhibitor of DNA damage induced by 500 micromol/L of H(2)O(2). The protective effect of VME against H(2)O(2)-induced oxidative damage on PC12 cells was investigated by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] reduction assay and lactate dehydrogenase (LDH) release assays. After 3 h of cell exposure to 500 micromol/L of H(2)O(2), a marked reduction in cell survival was observed. However, the reduction was significantly prevented by 100 and 250 microg/mL of VME. H(2)O(2) also induced severe apoptosis of the PC12 cells, which was indicated by Hoechst 33342 staining. Interestingly, the H(2)O(2)-stressed PC12 cells that were incubated with 100 and 250 microg/mL of VME had greatly suppressed apoptosis. The results suggest that VME could be a new antioxidant candidate against neuronal diseases.

  11. Comparison of DNA damage by methylmelamines and formaldehyde

    SciTech Connect

    Ross, W.E.; McMillan, D.R.; Ross, C.F.

    1981-07-01

    The cytotoxicity and DNA damaging activity of S9-activated hexamethylmelamine (HMM) and pentamethylmelamine (PMM) were compared with suspected active metabolites in mouse leukemia L1210 cells. Following treatment of L1210 cells with high concentrations of activated HMM and PMM, there were no DNA single-strand breaks or interstrand cross-links observed by DNA alkaline elution and only a low frequency of DNA-protein cross-links. Formaldehyde (FA) at nonlethal concentrations caused far greater DNA-protein cross-linking. The cytotoxicities of HMM and PMM were found unlikely to be related to extracellular or intracellular release of FA.

  12. LZ-106, a novel analog of enoxacin, inducing apoptosis via activation of ROS-dependent DNA damage response in NSCLCs.

    PubMed

    Yang, Lin; Yuan, Yinan; Fu, Chengyu; Xu, Xuefen; Zhou, Jieying; Wang, Shuhao; Kong, Lingyi; Li, Zhiyu; Guo, Qinglong; Wei, Libin

    2016-06-01

    Lung cancer, especially non-small-cell lung cancer (NSCLC), plays the leading role in cancer which is closely related to a myriad of fatal results. Unfortunately, current molecular mechanisms and clinical treatment of NSCLC still remain to be explored despite the fact that intensive investigations have been carried out in the last two decades. Recently, growing attention to finding exploitable sources of anticancer agents is refocused on quinolone compounds, an antibiotic with a long period of clinic application, for their remarkable cell-killing activity against not only bacteria, but eukaryotes as well. In this study, we found LZ-106, an analog of enoxacin, exhibiting potent inhibitory effects on NSCLC in both cultured cells and xenograft mouse model. We identified apoptosis-inducing action of LZ-106 in NSCLC cells through the mitochondrial and endoplasmic reticulum (ER)-stress apoptotic pathways via Annexin-V/PI double-staining assay, membrane potential detection, calcium level detection and the expression analysis of the key apoptotic proteins. Through comet assay, reactive oxygen species (ROS) detection, the expression analysis of DNA damage response (DDR) marker γ-H2AX and other DDR-related proteins, we also demonstrated that LZ-106 notably induced ROS overproduction and DDR. Interestingly, additional evidence in our findings revealed that DDR and apoptosis could be alleviated in the presence of ROS scavenger N-acetyl-cysteine (NAC), indicating ROS-dependent DDR involvement in LZ-106-induced apoptosis. Thus our data not only offered a new therapeutic candidate for NSCLC, but also put new insights into the pharmacological research of quinolones.

  13. DNA damage triggers genetic exchange in Helicobacter pylori.

    PubMed

    Dorer, Marion S; Fero, Jutta; Salama, Nina R

    2010-01-01

    Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen. PMID:20686662

  14. Persistent damage induces mitochondrial DNA degradation.

    PubMed

    Shokolenko, Inna N; Wilson, Glenn L; Alexeyev, Mikhail F

    2013-07-01

    Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6h after induction of mutant uracil-N-glycosylase and by 12h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and "foaming" of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.

  15. 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

  16. Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L.

    PubMed Central

    Panda, Brahma B.; Achary, V. Mohan M.

    2014-01-01

    In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa. PMID:24926302

  17. 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

  18. 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.

  19. 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.

  20. Age to survive: DNA damage and aging.

    PubMed

    Schumacher, Björn; Garinis, George A; Hoeijmakers, Jan H J

    2008-02-01

    Aging represents the progressive functional decline and increased mortality risk common to nearly all metazoans. Recent findings experimentally link DNA damage and organismal aging: longevity-regulating genetic pathways respond to the accumulation of DNA damage and other stress conditions and conversely influence the rate of damage accumulation and its impact for cancer and aging. This novel insight has emerged from studies on human progeroid diseases and mouse models that have deficient DNA repair pathways. Here we discuss a unified concept of an evolutionarily conserved 'survival' response that shifts the organism's resources from growth to maintenance as an adaptation to stresses, such as starvation and DNA damage. This shift protects the organism from cancer and promotes healthy aging. PMID:18192065

  1. In vitro packaging of damaged bacteriophage T7 DNA

    SciTech Connect

    Masker, W. E.; Kuemmerle, N. B.; Dodson, L. A.

    1980-01-01

    Experiments using in vitro packaging to monitor the biological activity of DNA recovered after in vitro repair, replication, and recombination reactions are described. These results suggest that the in vitro systems mimic the in vivo situation sufficiently well to allow generation (or restoration) of DNA molecules which can be encapsulated to form fully viable T7 phage particles. The in vitro packaging system has proved to be a convenient and relatively sensitive means for determining the amount of biological damage present in T7 DNA and for examining the response of various DNA metabolic systems to that damage.

  2. MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response.

    PubMed

    Mancini, F; Pieroni, L; Monteleone, V; Lucà, R; Fici, L; Luca, E; Urbani, A; Xiong, S; Soddu, S; Masetti, R; Lozano, G; Pontecorvi, A; Moretti, F

    2016-01-14

    The p53 inhibitor, MDM4 (MDMX) is a cytoplasmic protein with p53-activating function under DNA damage conditions. Particularly, MDM4 promotes phosphorylation of p53 at Ser46, a modification that precedes different p53 activities. We investigated the mechanism by which MDM4 promotes this p53 modification and its consequences in untransformed mammary epithelial cells and tissues. In response to severe DNA damage, MDM4 stimulates p53Ser46(P) by binding and stabilizing serine-threonine kinase HIPK2. Under these conditions, the p53-inhibitory complex, MDM4/MDM2, dissociates and this allows MDM4 to promote p53/HIPK2 functional interaction. Comparative proteomic analysis of DNA damage-treated cells versus -untreated cells evidenced a diffuse downregulation of proteins with anti-apoptotic activity, some of which were targets of p53Ser46(P)/HIPK2 repressive activity. Importantly, MDM4 depletion abolishes the downregulation of these proteins indicating the requirement of MDM4 to promote p53-mediated transcriptional repression. Consistently, MDM4-mediated HIPK2/p53 activation precedes HIPK2/p53 nuclear translocation and activity. Noteworthy, repression of these proteins was evident also in mammary glands of mice subjected to γ-irradiation and was significantly enhanced in transgenic mice overexpressing MDM4. This study evidences the flexibility of MDM2/MDM4 heterodimer, which allows the development of a positive activity of cytoplasmic MDM4 towards p53-mediated transcriptional function. Noteworthy, this activity uncovers coordinated repression of molecules with shared anti-apoptotic function which precedes active cell apoptosis and that are frequently overexpressed and/or markers of tumour phenotype in human cancer. PMID:25961923

  3. Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair

    PubMed Central

    Scott, Timothy L.; Rangaswamy, Suganya; Wicker, Christina A.

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. Recent Advances: In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. Critical Issues: The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. Future Directions: Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation. Antioxid. Redox Signal. 20, 708–726. PMID:23901781

  4. 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.

  5. Phosphorylated (pT371)TRF1 is recruited to sites of DNA damage to facilitate homologous recombination and checkpoint activation

    PubMed Central

    McKerlie, Megan; Walker, John R.; Mitchell, Taylor R. H.; Wilson, Florence R.; Zhu, Xu-Dong

    2013-01-01

    TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity. PMID:23997120

  6. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    PubMed Central

    Fasullo, Michael; Endres, Lauren

    2015-01-01

    Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases. PMID:25923076

  7. Oxidative stress and DNA damage in agricultural workers.

    PubMed

    Kisby, Glen E; Muniz, Juan F; Scherer, Jennifer; Lasarev, Michael R; Koshy, Mary; Kow, Yoke W; McCauley, Linda

    2009-01-01

    Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A pilot study of pesticide applicators and farm workers working in the fruit orchards of Oregon (i.e., apples, pears) was conducted to examine the relationship between organophosphate (OP) pesticide exposure and oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay) and serum analyzed for lipid peroxides (i.e., malondialdehyde [MDA]). Cellular DNA damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farm workers and applicators (p < .001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farm workers and applicators, respectively, than in controls. Serum MDA levels were 4.9 times and 24 times higher in farm workers and applicators, respectively, than in controls. DNA damage and oxidative DNA repair were significantly greater in lymphocytes from applicators and farm workers when compared with controls. A separate field study showed that DNA damage was also significantly greater (p < .001) in buccal cells (i.e., leukocytes) collected from migrant farm workers working with fungicides in the berry crops in Oregon. Markers of oxidative stress (i.e., reactive oxygen species, reduced levels of glutathione) and oxidative DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and cancer. PMID:19437279

  8. 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.

  9. [UV radiation, tanning and DNA damage].

    PubMed

    Koulu, Leena

    2014-01-01

    Excessive exposure to UV radiation is the most significant known risk factor for skin cancer. Solarium devices produce UVA radiation that is 5 to 10 times stronger than that produced by the sun. All wavelengths of UV radiation cause DNA damage to skin cells and cause tanning. Tanning protects skin cells from further damage. The DNA damages caused by UVA and UVB radiation, however, differ from each other. The protective capacity of tanning caused by UVA radiation seems to be lower than that caused by UVB radiation.

  10. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    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. PMID:25486562

  11. 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.

  12. Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2

    PubMed Central

    Quevedo, Oliver; García-Luis, Jonay; Matos-Perdomo, Emiliano; Aragón, Luis; Machín, Félix

    2012-01-01

    The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction. PMID:22363215

  13. White tea (Camellia sinensis) inhibits proliferation of the colon cancer cell line, HT-29, activates caspases and protects DNA of normal cells against oxidative damage.

    PubMed

    Hajiaghaalipour, Fatemeh; Kanthimathi, M S; Sanusi, Junedah; Rajarajeswaran, Jayakumar

    2015-02-15

    Tea (Camellia sinensis) is one of the most consumed beverages in the world. White tea is made from the buds and young leaves of the tea plant which are steamed and dried, whilst undergoing minimal oxidation. The MTT assay was used to test the extract on the effect of the proliferation of the colorectal cancer cell line, HT-29. The extract inhibited the proliferation of HT-29 cells with an IC50 of 87μg/ml. The extract increased the levels of caspase-3, -8, and -9 activity in the cells. DNA damage in 3T3-L1 normal cells was detected by using the comet assay. The extract protected 3T3-L1 cells against H2O2-induced DNA damage. The results from this study show that white tea has antioxidant and antiproliferative effects against cancer cells, but protect normal cells against DNA damage. Regular intake of white tea can help to maintain good health and protect the body against disease.

  14. Role of P-450 activity and glutathione levels in 1,2-dibromo-3-chloropropane tissue distribution, renal necrosis and in vivo DNA damage.

    PubMed

    Låg, M; Omichinski, J G; Søderlund, E J; Brunborg, G; Holme, J A; Dahl, J E; Nelson, S D; Dybing, E

    1989-06-16

    Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study. PMID:2734806

  15. Antiproliferative activity of goniothalamin enantiomers involves DNA damage, cell cycle arrest and apoptosis induction in MCF-7 and HB4a cells.

    PubMed

    Semprebon, Simone Cristine; Marques, Lilian Areal; D'Epiro, Gláucia Fernanda Rocha; de Camargo, Elaine Aparecida; da Silva, Glenda Nicioli; Niwa, Andressa Megumi; Macedo Junior, Fernando; Mantovani, Mário Sérgio

    2015-12-25

    (R)-goniothalamin (R-GNT) is a styryl lactone that exhibits antiproliferative property against several tumor cell lines. (S)-goniothalamin (S-GNT) is the synthetic enantiomer of R-GNT, and their biological properties are poorly understood. The aim of this study was to evaluate the antiproliferative mechanisms of (R)-goniothalamin and (S)-goniothalamin in MCF-7 breast cancer cells and HB4a epithelial mammary cells. To determine the mechanisms of cell growth inhibition, we analyzed the ability of R-GNT and S-GNT to induce DNA damage, cell cycle arrest and apoptosis. Moreover, the gene expression of cell cycle components, including cyclin, CDKs and CKIs, as well as of genes involved in apoptosis and the DNA damage response were evaluated. The natural enantiomer R-GNT proved more effective in both cell lines than did the synthetic enantiomer S-GNT, inhibiting cell proliferation via cell cycle arrest and apoptosis induction, likely in response to DNA damage. The cell cycle inhibition caused by R-GNT was mediated through the upregulation of CIP/KIP cyclin-kinase inhibitors and through the downregulation of cyclins and CDKs. S-GNT, in turn, was able to cause G0/G1 cell cycle arrest and DNA damage in MCF-7 cells and apoptosis induction only in HB4a cells. Therefore, goniothalamin presents potent antiproliferative activity to breast cancer cells MCF-7. However, exposure to goniothalamin brings some undesirable effects to non-tumor cells HB4a, including genotoxicity and apoptosis induction.

  16. DNA Damage Protecting Activity and Free Radical Scavenging Activity of Anthocyanins from Red Sorghum (Sorghum bicolor) Bran

    PubMed Central

    Devi, P. Suganya; Kumar, M. Saravana; Das, S. Mohan

    2012-01-01

    There is increasing interest in natural food colorants like carotenoids and anthocyanins with functional properties. Red sorghum bran is known as a rich source for anthocyanins. The anthocyanin contents extracted from red sorghum bran were evaluated by biochemical analysis. Among the three solvent system used, the acidified methanol extract showed a highest anthocyanin content (4.7 mg/g of sorghum bran) followed by methanol (1.95 mg/g) and acetone (1 mg/g). Similarly, the highest total flavonoids (143 mg/g) and total phenolic contents (0.93 mg/g) were obtained in acidified methanol extracts than methanol and acetone extracts. To study the health benefits of anthocyanin from red sorghum bran, the total antioxidant activity was evaluated by biochemical and molecular methods. The highest antioxidant activity was observed in acidified methanol extracts of anthocyanin in dose-dependent manner. The antioxidant activity of the red sorghum bran was directly related to the total anthocyanin found in red sorghum bran. PMID:22400119

  17. Regulation of the DNA damage response by ubiquitin conjugation

    PubMed Central

    Brinkmann, Kerstin; Schell, Michael; Hoppe, Thorsten; Kashkar, Hamid

    2015-01-01

    In response to DNA damage, cells activate a highly conserved and complex kinase-based signaling network, commonly referred to as the DNA damage response (DDR), to safeguard genomic integrity. The DDR consists of a set of tightly regulated events, including detection of DNA damage, accumulation of DNA repair factors at the site of damage, and finally physical repair of the lesion. Upon overwhelming damage the DDR provokes detrimental cellular actions by involving the apoptotic machinery and inducing a coordinated demise of the damaged cells (DNA damage-induced apoptosis, DDIA). These diverse actions involve transcriptional activation of several genes that govern the DDR. Moreover, recent observations highlighted the role of ubiquitylation in orchestrating the DDR, providing a dynamic cellular regulatory circuit helping to guarantee genomic stability and cellular homeostasis (Popovic et al., 2014). One of the hallmarks of human cancer is genomic instability (Hanahan and Weinberg, 2011). Not surprisingly, deregulation of the DDR can lead to human diseases, including cancer, and can induce resistance to genotoxic anti-cancer therapy (Lord and Ashworth, 2012). Here, we summarize the role of ubiquitin-signaling in the DDR with special emphasis on its role in cancer and highlight the therapeutic value of the ubiquitin-conjugation machinery as a target in anti-cancer treatment strategy. PMID:25806049

  18. Circadian Modulation of 8-Oxoguanine DNA Damage Repair

    PubMed Central

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  19. The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

    SciTech Connect

    Yoon, Jung-Hoon; Qiu Junzhuan; Cai Sheng; Chen Yuan; Cheetham, Michael E.; Shen Binghui; Pfeifer, Gerd P. . E-mail: gpfeifer@coh.org

    2006-05-01

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.

  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. Parvovirus infection-induced DNA damage response

    PubMed Central

    Luo, Yong; Qiu, Jianming

    2014-01-01

    Parvoviruses are a group of small DNA viruses with ssDNA genomes flanked by two inverted terminal structures. Due to a limited genetic resource they require host cellular factors and sometimes a helper virus for efficient viral replication. Recent studies have shown that parvoviruses interact with the DNA damage machinery, which has a significant impact on the life cycle of the virus as well as the fate of infected cells. In addition, due to special DNA structures of the viral genomes, parvoviruses are useful tools for the study of the molecular mechanisms underlying viral infection-induced DNA damage response (DDR). This review aims to summarize recent advances in parvovirus-induced DDR, with a focus on the diverse DDR pathways triggered by different parvoviruses and the consequences of DDR on the viral life cycle as well as the fate of infected cells. PMID:25429305

  2. Assessment of free radical scavenging potential and oxidative DNA damage preventive activity of Trachyspermum ammi L. (carom) and Foeniculum vulgare Mill. (fennel) seed extracts.

    PubMed

    Goswami, Nandini; Chatterjee, Sreemoyee

    2014-01-01

    Oxidation of biomolecules such as carbohydrates, proteins, lipids, and nucleic acids results in generation of free radicals in an organism which is the major cause of onset of various degenerative diseases. Antioxidants scavenge these free radicals, thereby protecting the cell from damage. The present study was designed to examine the free radical scavenging potential and oxidative DNA damage preventive activity of traditionally used spices Trachyspermum ammi L. (carom) and Foeniculum vulgare Mill. (fennel). The aqueous, methanolic, and acetonic extracts of T. ammi and F. vulgare seeds were prepared using soxhlet extraction assembly and subjected to qualitative and quantitative estimation of phytochemical constituents. Free radical scavenging potential was investigated using standard methods, namely, DPPH radical scavenging assay and ferric reducing antioxidant power assay along with the protection against oxidative DNA damage. The results stated that acetonic seed extracts (AAcSE and FAcSE) of both the spices possessed comparatively high amount of total phenolics whereas methanolic seed extracts (AMSE and FMSE) were found to have highest amount of total flavonoids. At 1 mg/mL concentration, highest DPPH radical scavenging activity was shown by FMSE (96.2%), AAcSE was recorded with highest FRAP value (2270.27 ± 0.005 μmol/L), and all the seed extracts have been shown to mitigate the damage induced by Fenton reaction on calf thymus DNA. Therefore, the study suggests that T. ammi and F. vulgare seed extracts could contribute as a highly significant bioresource of antioxidants to be used in our day-to-day life and in food and pharmaceutical industry.

  3. Assessment of Free Radical Scavenging Potential and Oxidative DNA Damage Preventive Activity of Trachyspermum ammi L. (Carom) and Foeniculum vulgare Mill. (Fennel) Seed Extracts

    PubMed Central

    2014-01-01

    Oxidation of biomolecules such as carbohydrates, proteins, lipids, and nucleic acids results in generation of free radicals in an organism which is the major cause of onset of various degenerative diseases. Antioxidants scavenge these free radicals, thereby protecting the cell from damage. The present study was designed to examine the free radical scavenging potential and oxidative DNA damage preventive activity of traditionally used spices Trachyspermum ammi L. (carom) and Foeniculum vulgare Mill. (fennel). The aqueous, methanolic, and acetonic extracts of T. ammi and F. vulgare seeds were prepared using soxhlet extraction assembly and subjected to qualitative and quantitative estimation of phytochemical constituents. Free radical scavenging potential was investigated using standard methods, namely, DPPH radical scavenging assay and ferric reducing antioxidant power assay along with the protection against oxidative DNA damage. The results stated that acetonic seed extracts (AAcSE and FAcSE) of both the spices possessed comparatively high amount of total phenolics whereas methanolic seed extracts (AMSE and FMSE) were found to have highest amount of total flavonoids. At 1 mg/mL concentration, highest DPPH radical scavenging activity was shown by FMSE (96.2%), AAcSE was recorded with highest FRAP value (2270.27 ± 0.005 μmol/L), and all the seed extracts have been shown to mitigate the damage induced by Fenton reaction on calf thymus DNA. Therefore, the study suggests that T. ammi and F. vulgare seed extracts could contribute as a highly significant bioresource of antioxidants to be used in our day-to-day life and in food and pharmaceutical industry. PMID:25143939

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

    PubMed

    Oh, D H; King, B A; Boxer, S G; Hanawalt, P C

    2001-09-25

    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

  5. 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

  6. DNA Damage Response Genes and the Development of Cancer Metastasis

    PubMed Central

    Broustas, Constantinos G.; Lieberman, Howard B.

    2014-01-01

    DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

  7. DNA damage phenotype and prostate cancer risk

    PubMed Central

    Kosti, O.; Goldman, L.; Saha, D.T.; Orden, R.A.; Pollock, A.J.; Madej, H.L.; Hsing, A.W.; Chu, L.W.; Lynch, J.H.; Goldman, R.

    2010-01-01

    The capacity of an individual to process DNA damage is considered a crucial factor in carcinogenesis. The comet assay is a phenotypic measure of the combined effects of sensitivity to a mutagen exposure and repair capacity. In this paper, we evaluate the association of the DNA repair kinetics, as measured by the comet assay, with prostate cancer risk. In a pilot study of 55 men with prostate cancer, 53 men without the disease, and 71 men free of cancer at biopsy, we investigated the association of DNA damage with prostate cancer risk at early (0-15 min) and later (15-45 min) stages following gamma-radiation exposure. Although residual damage within 45 min was the same for all groups (65% of DNA in comet tail disappeared), prostate cancer cases had a slower first phase (38% vs 41%) and faster second phase (27% vs 22%) of the repair response compared to controls. When subjects were categorized into quartiles, according to efficiency of repairing DNA damage, high repair-efficiency within the first 15 min after exposure was not associated with prostate cancer risk while higher at the 15-45 min period was associated with increased risk (OR for highest-to-lowest quartiles = 3.24, 95% CI=0.98-10.66, p-trend =0.04). Despite limited sample size, our data suggest that DNA repair kinetics marginally differ between prostate cancer cases and controls. This small difference could be associated with differential responses to DNA damage among susceptible individuals. PMID:21095241

  8. The role of oxidative and conjugative pathways in the activation of 1,2-dibromo-3-chloropropane to DNA-damaging products in rat testicular cells.

    PubMed

    Omichinski, J G; Brunborg, G; Holme, J A; Søderlund, E J; Nelson, S D; Dybing, E

    1988-07-01

    The ability of 1,2-dibromo-3-chloropropane (DBCP), several methylated analogs of DBCP and perdeuterated DBCP (DBCP-D5) to cause DNA damage in isolated testicular cells from rats was measured by the alkaline elution technique. Of the methylated analogs studied, only the C3-methyl analog was capable of causing significant DNA damage at concentrations of 0-50 microM. In both time- (0-60 min) and concentration- (0-10 microM) dependent experiments, the testicular cell DNA damage caused by the perdeuterated analog of DBCP closely mimicked the damage resulting from DBCP itself. The lack of an isotope effect between DBCP-D5 and DBCP strongly suggests that metabolism via a cytochrome P-450-dependent pathway is not involved in the DNA-damaging effects of DBCP in rat testicular cells. In contrast, preincubation for 1 hr with diethylmaleate (DEM) inhibited DBCP-induced (10 microM) DNA damage in a concentration-dependent manner (0-500 microM DEM). The decrease in testicular DNA damage was proportional to the decrease in cellular nonprotein sulfhydryl levels. Similarly, it was shown that 1,2-dibromoethane (EDB), a structurally related halogenated alkane, produced DNA damage in isolated testicular cells in both a time- (0-60 min) and concentration- (0-600 microM) dependent fashion. The DNA damage produced by EDB (600 microM) was also inhibited by pretreatment of testicular cells with DEM (1 mM). The testicular genotoxicity induced by EDB is thought to involve its initial conjugation to glutathione and the subsequent formation of a reactive episulfonium ion. The data presented indicate that similar events may be occurring in DBCP-induced DNA damage in rat testicular cells. PMID:3393142

  9. 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.

  10. Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity.

    PubMed

    Slyskova, Jana; Lorenzo, Yolanda; Karlsen, Anette; Carlsen, Monica H; Novosadova, Vendula; Blomhoff, Rune; Vodicka, Pavel; Collins, Andrew R

    2014-04-01

    The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity. Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways. Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p=0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by β-cryptoxanthin and β-tocopherol (p<0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p=0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and α-carotene (p<0.05). We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity. PMID:24674629

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:26906421

  12. 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. PMID:26906421

  13. Cu(II)-coumestrol interaction leads to ROS-mediated DNA damage and cell death: a putative mechanism for anticancer activity.

    PubMed

    Zafar, Atif; Singh, Swarnendra; Naseem, Imrana

    2016-07-01

    Phytoestrogens have attracted considerable interest as natural alternatives to hormone replacement therapy and their potential as cancer therapeutic agents. Among phytoestrogens, coumestrol has shown multipharmacological properties such as antiinflammatory, neuroprotective, osteoblastic differentiation and anticancer. Though several studies have described anticancer effects of coumestrol, a clear underlying molecular mechanism has not been elucidated. Unlike normal cells, cancer cells contain elevated copper levels that play an integral role in angiogenesis. Copper is an important metal ion associated with the chromatin DNA, particularly with guanine. Thus, targeting copper in cancer cells can serve as effective anticancer strategy. Using human peripheral lymphocytes, we assessed lipid peroxidation, protein carbonylation, reactive oxygen species (ROS) generation, DNA damage and apoptosis by coumestrol in the presence of exogenously added Cu(II) in cells to simulate malignancy-like condition. Results showed that Cu(II)-coumestrol interaction leads to lipid peroxidation and protein carbonylation (markers of oxidative stress), DNA fragmentation and apoptosis in treated lymphocytes. Further, incubation of lymphocytes with ROS scavengers and membrane-permeant copper chelator, neocuproine, resulted in inhibition of DNA damage and apoptosis. This suggests that coumestrol engages in redox cycling of Cu(II) to generate ROS that leads to DNA fragmentation and apoptosis. In conclusion, this is the first report showing that coumestrol targets cellular copper to induce prooxidant death in malignant cells. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of coumestrol. These findings will provide significant insights into the development of new chemical molecules with better copper-chelating and prooxidant properties against cancer cells. PMID:27260464

  14. IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation*

    PubMed Central

    Hwang, Byounghoon; McCool, Kevin; Wan, Jun; Wuerzberger-Davis, Shelly M.; Young, Edmond W. K.; Choi, Eun Young; Cingolani, Gino; Weaver, Beth A.; Miyamoto, Shigeki

    2015-01-01

    Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and β family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin β family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response. PMID:26060253

  15. IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation.

    PubMed

    Hwang, Byounghoon; McCool, Kevin; Wan, Jun; Wuerzberger-Davis, Shelly M; Young, Edmond W K; Choi, Eun Young; Cingolani, Gino; Weaver, Beth A; Miyamoto, Shigeki

    2015-07-17

    Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and β family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin β family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.

  16. p28-Mediated Activation of p53 in G2-M Phase of the Cell Cycle Enhances the Efficacy of DNA Damaging and Antimitotic Chemotherapy.

    PubMed

    Yamada, Tohru; Das Gupta, Tapas K; Beattie, Craig W

    2016-04-15

    p28 is an anionic cell-penetrating peptide of 28 amino acids that activates wild-type and mutated p53, leading subsequently to selective inhibition of CDK2 and cyclin A expression and G2-M cell-cycle arrest. In this study, we investigated the cytotoxic effects of p28 treatment alone and in combination with DNA-damaging and antimitotic agents on human cancer cells. p28 enhanced the cytotoxic activity of lower concentrations (IC20-50) of DNA-damaging drugs (doxorubicin, dacarbazine, temozolamide) or antimitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells expressing wild-type or mutated p53. Mechanistic investigations revealed that p28 induced a post-translational increase in the expression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at the G2-M phase. The enhanced activity of these anticancer agents in combination with p28 was facilitated through the p53/p21/CDK2 pathway. Taken together, these results highlight a new approach to maximize the efficacy of chemotherapeutic agents while reducing dose-related toxicity. Cancer Res; 76(8); 2354-65. ©2016 AACR. PMID:26921335

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

    PubMed Central

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

    2014-01-01

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

  18. Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

    PubMed Central

    Chitikova, Zhanna V; Gordeev, Serguei A; Bykova, Tatiana V; Zubova, Svetlana G; Pospelov, Valery A; Pospelova, Tatiana V

    2014-01-01

    Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G2/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers. PMID:24626185

  19. DNA Damage and Repair in Eukaryotic Cells

    PubMed Central

    Painter, R. B.

    1974-01-01

    Damage in DNA after irradiation can be classified into five kinds: base damage, single-strand breaks, double-strand breaks, DNA–DNA cross-linking, and DNA-protein cross-linking. Of these, repair of base damage is the best understood. In eukaryotes, at least three repair systems are known that can deal with base damage: photoreactivation, excision repair, and post-replication repair. Photoreactivation is specific for UV-induced damage and occurs widely throughout the biosphere, although it seems to be absent from placental mammals. Excision repair is present in prokaryotes and in animals but does not seem to be present in plants. Post-replication repair is poorly understood. Recent reports indicate that growing points in mammalian DNA simply skip past UV-induced lesions, leaving gaps in newly made DNA that are subsequently filled in by de novo synthesis. Evidence that this concept is oversimplified or incorrect is presented.—Single-strand breaks are induced by ionizing radiation but most cells can rapidly repair most or all of them, even after supralethal doses. The chemistry of the fragments formed when breaks are induced by ionizing radiation is complex and poorly understood. Therefore, the intermediate steps in the repair of single-strand breaks are unknown. Double-strand breaks and the two kinds of cross-linking have been studied very little and almost nothing is known about their mechanisms for repair.—The role of mammalian DNA repair in mutations is not known. Although there is evidence that defective repair can lead to cancer and/or premature aging in humans, the relationship between the molecular defects and the diseased state remains obscure. PMID:4442699

  20. UV damage in DNA promotes nucleosome unwrapping.

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2010-08-20

    The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased "intrinsic exposure" of nucleosome-associated DNA lesions in chromatin to repair proteins. PMID:20562439

  1. Genotoxic stress in plants: shedding light on DNA damage, repair and DNA repair helicases.

    PubMed

    Tuteja, Narendra; Ahmad, Parvaiz; Panda, Brahma B; Tuteja, Renu

    2009-01-01

    Plant cells are constantly exposed to environmental agents and endogenous processes that inflict damage to DNA and cause genotoxic stress, which can reduce plant genome stability, growth and productivity. Plants are most affected by solar UV-B radiation, which damage the DNA by inducing the formation of two main UV photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Reactive oxygen species (ROS) are also generated extra- or intra-cellularly, which constitute yet another source of genotoxic stress. As a result of this stress, the cellular DNA-damage responses (DDR) are activated, which transiently arrest the cell cycle and allow cells to repair DNA before proceeding into mitosis. DDR requires the activation of Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) genes, which regulate the cell cycle and transmit the damage signals to downstream effectors of cell-cycle progression. Since genomic protection and stability are fundamental to ensure and sustain plant diversity and productivity, therefore, repair of DNA damages is essential. In plants the bulky DNA lesions, CPDs and 6-4PPs, are repaired by a simple and error-free mechanism: photoreactivation, which is a light-dependent mechanism and requires CPD or 6-4PP specific photolyases. In addition to this direct repair process, the plants also have sophisticated light-independent general repair mechanisms, such as the nucleotide excision repair (NER) and base excision repair (BER). The completed plant genome sequences reveal that most of the genes involved in NER and BER are present in higher plants, which suggests that the network of in-built DNA-damage repair mechanisms is conserved. This article describes the insight underlying the DNA damage and repair pathways in plants. The comet assay to measure the DNA damage and the role of DNA repair helicases such as XPD and XPB are also covered.

  2. Oxidation of DNA: damage to nucleobases.

    PubMed

    Kanvah, Sriram; Joseph, Joshy; Schuster, Gary B; Barnett, Robert N; Cleveland, Charles L; Landman, Uzi

    2010-02-16

    All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and

  3. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    PubMed Central

    Edelbrock, Michael A.; Kaliyaperumal, Saravanan; Williams, Kandace J.

    2013-01-01

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch Syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O6meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6. PMID:23391514

  4. A novel bisindole-PBD conjugate causes DNA damage induced apoptosis via inhibition of DNA repair pathway

    PubMed Central

    Sarma, Pranjal; Ramaiah, M Janaki; Kamal, Ahmed; Bhadra, Utpal; Bhadra, Manika Pal

    2014-01-01

    DNA damage response (DDR) that includes cell cycle check points, DNA repair, apoptosis, and senescence is intimately linked with cancer. It shields an organism against cancer development when genomic integrity fails. DNA repair pathways protect the cells from tumor progression caused as a result of DNA damage induced by irradiation or due to chemotherapeutic treatment. Many promising anticancer agents have been identified that target specific DNA repair pathways in response to DNA damage thereby leading to apoptosis. Here we identified a novel bisindole-PBD conjugate that possess potent anticancer activity in breast cancer cells. Further studies aimed at understanding the mechanism of action of the molecule showed its role in DNA damage induced apoptosis via inhibition of DNA repair pathway. Trypan blue and BrdU assay exhibited a dose-dependent effect. Single-stranded DNA damage was observed by COMET assay. In addition DNA damage induced ROS generation with simultaneous activation of ATM and ATR upon compound treatment was observed. Further downregulation of Bcl-XL and activation of Bax showed DNA damage induced apoptosis in MCF-7 and MDAMB-231 cells. In conclusion, it can be summarized that bisindole-PBD conjugate induces DNA damage in a dose dependent (2, 4, and 8 μM) manner by inhibiting the DNA repair genes. PMID:25010292

  5. 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...

  6. Assaying DNA damage in hippocampal neurons using the comet assay.

    PubMed

    Nowsheen, Somaira; Xia, Fen; Yang, Eddy S

    2012-12-19

    A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique. The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell (1,2). Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

  7. Oxidative DNA Damage and Nucleotide Excision Repair

    PubMed Central

    Melis, Joost P.M.; Luijten, Mirjam

    2013-01-01

    Abstract Significance: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. Recent Advances: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. Critical Issues: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Future Directions: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues. Antioxid. Redox Signal. 18, 2409–2419. PMID:23216312

  8. Actinic DNA damage and the pathogenesis of cutaneous malignant melanoma.

    PubMed

    Ross, P M; Carter, D M

    1989-05-01

    The near epidemic of melanoma and non-melanoma skin cancer in the United States and certain other industrialized nations is attributable to cutaneous exposure to sunlight more than to any other factor. Chronic exposure to UV irradiation and a high total cumulative dose may be less deleterious than are periodic bursts of large amounts of sun exposure leading to severe sunburn. Such an exposure pattern is characteristic of individuals such as office workers whose outdoor activities are irregular rather than daily, as with farmers or fisherman. Although UV irradiation is injurious to many cellular elements, the mechanisms underlying UV-mediated skin cancer are thought to be most likely related to DNA damage to cutaneous cells. Various types of UV-induced DNA damage have been identified, and they differ in biologic significance. Damage which is apt to be most cytotoxic is probably less effective as an inducer of skin cancer than is more subtle damage, which is tolerated but can initiate malignant transformation. Repair of DNA damage involves specific cellular activities which vary in their effectiveness in restoring cutaneous cell function to normal. Other biologic effects of UV irradiation may contribute to the development of skin cancer through effects on such defenses as pigmentation and the immune response. Sun-induced damage to DNA, however, is apparently necessary. Biologic consequences of dangerous environmental exposure to UV irradiation can be modulated by changes in life-style, the depth of the ozone layer, use of sunscreens, and possibly by hormones or their synthetic analogs.

  9. The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes.

    PubMed

    van Oosterwijk, M F; Versteeg, A; Filon, R; van Zeeland, A A; Mullenders, L H

    1996-08-01

    Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells

  10. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    PubMed Central

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  11. Damage and repair of ancient DNA.

    PubMed

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-04-01

    Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes such as extinct horses, cave bears, the marsupial wolf, the moa, and Neanderthal. In the past few years, this technology has been extended to the study of infectious disease in ancient Egyptian and South American mummies, the dietary habits of ancient animals, and agricultural practices and population dynamics of early native Americans. Hence, ancient DNA contains information pertinent to numerous fields of study including evolution, population genetics, ecology, climatology, medicine, archeology, and behavior. The major obstacles to the study of aDNA are its extremely low yield, contamination with modern DNA, and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived from limited sources. In addition, we will present some of our own preliminary and published data on mechanisms of DNA degradation and some speculative thoughts on strategies for repair and restoration of aDNA.

  12. Oxidative DNA damage in relation to nutrition.

    PubMed

    Krajcovicová-Kudlácková, M; Dusinská, M

    2004-01-01

    Oxidative DNA damage in humans could arise also from incorrect nutritional habit and life style. DNA strand breaks with apurinic/apyrimidinic sites, oxidized purines and oxidized pyrimidines were assessed in 24 subjectively healthy vegetarians (plant food, dairy products, eggs) and compared with 24 non-vegetarians (traditional diet, general population). DNA strand breaks + oxidized purines are significantly reduced in vegetarians (p<0.05), DNA strand breaks are nonsignificantly decreased. The sufficient antioxidative status (overthreshold values of natural essential antioxidants, which mean a reduced risk of free radical disease) is crucial in free radical defense. Intake of protective food commodities (fruit, vegetables, dark grain products, grain sprouts, oil seeds) is significantly higher in vegetarians. Alternative nutrition subjects have a significantly increased plasma levels of vitamin C, vitamin E, beta-carotene with high incidence of overthreshold values (92% vs. 42% - vitamin C, 67% vs. 33% - vitamin E, 67% vs. 17% - beta-carotene). There is recorded a significant inverse linear correlation between values of DNA strand breaks + oxidized purines and vitamin C or beta-carotene levels (p<0.01, p<0.05). Vegetarian diet is significantly more rich source of antioxidants. The results of reduced endogenous DNA damage and higher antioxidative status in vegetarians document that a correct vegetarian nutrition might represent an effective cancer prevention.

  13. 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.

  14. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds

    PubMed Central

    Sitarek, Przemysław; Skała, Ewa; Wysokińska, Halina; Wielanek, Marzena; Szemraj, Janusz; Toma, Monika; Śliwiński, Tomasz

    2016-01-01

    Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx). LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin), and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid). The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx) regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds. PMID:26788249

  15. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds.

    PubMed

    Sitarek, Przemysław; Skała, Ewa; Wysokińska, Halina; Wielanek, Marzena; Szemraj, Janusz; Toma, Monika; Śliwiński, Tomasz

    2016-01-01

    Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx). LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin), and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid). The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx) regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

  16. Diphenylarsinic acid, a chemical warfare-related neurotoxicant, promotes liver carcinogenesis via activation of aryl hydrocarbon receptor signaling and consequent induction of oxidative DNA damage in rats.

    PubMed

    Wei, Min; Yamada, Takanori; Yamano, Shotaro; Kato, Minoru; Kakehashi, Anna; Fujioka, Masaki; Tago, Yoshiyuki; Kitano, Mistuaki; Wanibuchi, Hideki

    2013-11-15

    Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes in the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies.

  17. The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage.

    PubMed

    Heidor, Renato; Furtado, Kelly Silva; Ortega, Juliana Festa; de Oliveira, Tiago Franco; Tavares, Paulo Eduardo Latorre Martins; Vieira, Alessandra; Miranda, Mayara Lilian Paulino; Purgatto, Eduardo; Moreno, Fernando Salvador

    2014-04-15

    The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200mg/100g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p<0.05) as well as the ACF with ≥4 crypts (p<0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p<0.05) and reduced DNA damage (p<0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p<0.05). TB administration resulted in increased colonic tissue concentrations of BA (p<0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB.

  18. Rose myrtle (Rhodomyrtus tomentosa) extract and its component, piceatannol, enhance the activity of DNA polymerase and suppress the inflammatory response elicited by UVB‑induced DNA damage in skin cells.

    PubMed

    Shiratake, Sawako; Nakahara, Tatsuo; Iwahashi, Hiroyasu; Onodera, Takefumi; Mizushina, Yoshiyuki

    2015-10-01

    A number of naturally occurring agents are hypothesized to protect against ultraviolet (UV)‑induced skin damage. The present study screened >50 plant extracts for inhibitors of UVB‑induced cytotoxicity, using cultured normal human epidermal keratinocytes (NHEK), and identified that the fruit of rose myrtle (Rhodomyrtus tomentosa) was the most marked inhibitor of cell death. The protective effect of rose myrtle extract and the two key components, piceatannol and piceatannol‑4'‑O‑β‑D‑glucopyranoside, on UVB‑induced damage and inflammation in cultured NHEK was investigated. The 80% ethanol extract from rose myrtle fruit with piceatannol exhibited protection of UVB‑induced cytotoxicity in NHEK; however, piceatannol‑4'‑O‑β‑D‑glucopyranoside exhibited no protection, as determined by a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. This extract and piceatannol reduced the production of UVB‑induced cyclobutane pyrimidine dimers and enhanced the cellular enzyme activity of the DNA polymerases in UVB‑irradiated NHEK, suggesting that UVB‑stimulated DNA damage was repaired by the polymerases. In addition, the secretion of prostaglandin E2, which is an inflammatory mediator, was decreased. These results indicated that rose myrtle fruit extract and its key constituent, piceatannol, are potential photoprotective candidates for UV‑induced skin damage.

  19. Rose myrtle (Rhodomyrtus tomentosa) extract and its component, piceatannol, enhance the activity of DNA polymerase and suppress the inflammatory response elicited by UVB‑induced DNA damage in skin cells.

    PubMed

    Shiratake, Sawako; Nakahara, Tatsuo; Iwahashi, Hiroyasu; Onodera, Takefumi; Mizushina, Yoshiyuki

    2015-10-01

    A number of naturally occurring agents are hypothesized to protect against ultraviolet (UV)‑induced skin damage. The present study screened >50 plant extracts for inhibitors of UVB‑induced cytotoxicity, using cultured normal human epidermal keratinocytes (NHEK), and identified that the fruit of rose myrtle (Rhodomyrtus tomentosa) was the most marked inhibitor of cell death. The protective effect of rose myrtle extract and the two key components, piceatannol and piceatannol‑4'‑O‑β‑D‑glucopyranoside, on UVB‑induced damage and inflammation in cultured NHEK was investigated. The 80% ethanol extract from rose myrtle fruit with piceatannol exhibited protection of UVB‑induced cytotoxicity in NHEK; however, piceatannol‑4'‑O‑β‑D‑glucopyranoside exhibited no protection, as determined by a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. This extract and piceatannol reduced the production of UVB‑induced cyclobutane pyrimidine dimers and enhanced the cellular enzyme activity of the DNA polymerases in UVB‑irradiated NHEK, suggesting that UVB‑stimulated DNA damage was repaired by the polymerases. In addition, the secretion of prostaglandin E2, which is an inflammatory mediator, was decreased. These results indicated that rose myrtle fruit extract and its key constituent, piceatannol, are potential photoprotective candidates for UV‑induced skin damage. PMID:26239705

  20. The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage

    SciTech Connect

    Heidor, Renato; Furtado, Kelly Silva; Ortega, Juliana Festa; Oliveira, Tiago Franco de; Tavares, Paulo Eduardo Latorre Martins; Vieira, Alessandra; Miranda, Mayara Lilian Paulino; Purgatto, Eduardo; Moreno, Fernando Salvador

    2014-04-15

    The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200 mg/100 g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100 g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p < 0.05) as well as the ACF with ≥ 4 crypts (p < 0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p < 0.05) and reduced DNA damage (p < 0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p < 0.05). TB administration resulted in increased colonic tissue concentrations of BA (p < 0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB. - Highlights: • Tributyrin is a chemopreventive agent for rat colon aberrant crypt foci. • Tributyrin increased apoptosis in an experimental rat colon carcinogenesis model. • Tributyrin treatment in a rat colon carcinogenesis model decreased DNA damage. • Tributyrin treatment induced H3K9 acetylation in a rat colon carcinogenesis model.

  1. 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

  2. Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1.

    PubMed

    Brinkmeyer, Megan K; David, Sheila S

    2015-10-01

    MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.

  3. An increase of oxidised nucleotides activates DNA damage checkpoint pathway that regulates post-embryonic development in Caenorhabditis elegans.

    PubMed

    Sanada, Yu; Zhang-Akiyama, Qiu-Mei

    2014-03-01

    8-Oxo-dGTP, an oxidised form of dGTP generated in the nucleotide pool, can be incorporated opposite adenine or cytosine in template DNA, which can in turn induce mutations. In this study, we identified a novel MutT homolog (NDX-2) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. In addition, we found that NDX-1, NDX-2 and NDX-4 proteins have 8-oxo-GTPase or 8-oxo-GDPase activity. The sensitivity of ndx-2 knockdown C. elegans worms to methyl viologen and menadione bisulphite was increased compared with that of control worms. This sensitivity was rescued by depletion of chk-2 and clk-2, suggesting that growth of the worms is regulated by the checkpoint pathway in response to the accumulation of oxidised nucleotides. Moreover, we found that the sensitivity to menadione bisulphite of ndx-1 and ndx-2-double knockdown worms was enhanced by elimination of XPA-1, a factor involved in nucleotide excision repair. The rescue effect by depletion of chk-2 and clk-2 was limited in the xpa-1 mutant, suggesting that the chk-2 and clk-2 checkpoint pathway is partially linked to the function of XPA-1.

  4. The dynamic behavior of Ect2 in response to DNA damage

    PubMed Central

    He, Dan; Xiang, Jinnan; Li, Baojie; Liu, Huijuan

    2016-01-01

    Ect2 is a BRCT-containing guanidine exchange factor for Rho GTPases. It is essential for cytokinesis and is also involved in tumorigenesis. Since most BRCT-containing proteins are involved in DNA damage response and/or DNA repair, we tested whether Ect2 plays similar roles. We report that in primary mouse embryonic fibroblasts (MEFs), DNA damage quickly led to Ect2 relocalization to the chromatin and DNA damage foci-like structures. Ect2 knockdown did not affect foci localization of γH2AX, TopBP1, or Brca1, or activation of Atm, yet it impeded p53 Ser15 phosphorylation and activation, and resulted in defects in apoptosis and activation of S and G2/M checkpoints in response to DNA damage. These results suggest that Ect2 plays a role in DNA damage response. Interestingly, Ect2 is down-regulated at late stages of DNA damage response. Although p53 and E2F1 have been shown to regulate Ect2 transcription, DNA damage-induced Ect2 down-regulation occurred in p53−/− or Atm−/− MEFs and E2F1 knockdown cells. Instead, DNA damage-induced Ect2 down-regulation is mainly attributable to decreased protein stability. Like Ect2 knockdown, Ect2 destabilization may help the cell to recover from DNA damage response. These results suggest that Ect2 plays roles in multiple aspects of DNA damage response. PMID:27074761

  5. Turning off the G2 DNA damage checkpoint

    PubMed Central

    Calonge, Teresa M.; O'Connell, Matthew J.

    2008-01-01

    In response to DNA damage, cells activate checkpoints to delay cell cycle progression and allow time for completion of DNA repair before commitment to S-phase or mitosis. During G2, many proteins collaborate to activate Chk1, an effector protein kinase that ensures the mitotic cyclin-dependent kinase remains in an inactive state. This checkpoint is ancient in origin and highly conserved from fission yeast to humans. Work from many groups has led to a detailed description of the spatiotemporal control of signaling events leading to Chk1 activation. However, to survive DNA damage in G2, the checkpoint must be inactivated to allow resumption of cell cycling and entry into mitosis. Though only beginning to be understood, here we review current data regarding checkpoint termination signals acting on Chk1 and its' upstream regulators. PMID:17851138

  6. Mechanistic studies of copper(II)-aminoglycoside mediated DNA damage and magnesium catalyzed nuclease activity of hammerhead ribozyme

    NASA Astrophysics Data System (ADS)

    Patwardhan, Anjali A.

    The antibacterial activity of aminoglycosides stems from their high affinity binding to the 16S rRNA in bacteria resulting in inhibition of protein synthesis. Used to treat acute bacterial infections these antibiotics have limited applications due to their high dosage requirements and the emergence of resistant strains. We have synthesized and characterized Cu(II) derivatives of the aminoglycosides, kanamycin A, tobramycin, neamine, kanamycin B, neomycin B, and paromomycin. The first three exhibit preferential and tight binding to Cu(II) as against neomycin B and kanamycin B and paromomycin. EPR of frozen solutions and UV-visible spectroscopy suggest a change in geometry around the Cu(II) but the stabilities of the complexes in water differ. These copper derivatives efficiently cleave plasmid DNA at micromolar concentrations (hydrolytic) and at nanomolar concentrations in the presence co-reactants like hydrogen peroxide or ascorbic acid. Hydrolysis is multi turnover and exhibits Michelis-Menten kinetics with enzyme-like behavior whereas oxidative cleavage is highly specific with C-4' H abstraction resulting in characteristic base propenal and nucleotide base products. Hydroxyl radicals generated are copper based and are generated in close proximity of the substrate. Hammerhead ribozymes are selectively hydrolyzed in the presence of divalent ions with Mg2+ being the metal ion of choice in vivo . Our studies with complex ions like cobalt hexaammine and fac-triamminetriaquochromium(III) establish outer sphere interactions of Mg2+ with the hammerhead in the catalytic site. There are two sets of sites, one structural and one catalytic. Complex ions in the catalytic site and divalent ions in the structural site result in a slow but active hammerhead ribozyme suggesting that the complex ions are not inhibitory, contrary to what was suggested previously.

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

    PubMed Central

    Hamperl, Stephan; Cimprich, Karlene A.

    2014-01-01

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

  8. DNA damage, homology-directed repair, and DNA methylation.

    PubMed

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

    2007-07-01

    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, approximately 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, 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

  10. Clinical implications of sperm DNA damage.

    PubMed

    Lewis, Sheena E M; Simon, Luke

    2010-12-01

    Traditionally, the diagnosis of male infertility has relied upon microscopic assessment and biochemical assays to determine human semen quality. These tests are essential to provide the fundamental information on which clinicians base their initial diagnosis. However, none of these parameters addresses sperm function and their clinical value in predicting fertility is questionable. The advent of intracytoplasmic sperm injection (ICSI) has further reduced the significance and perceived need for sperm quality tests since ICSI requires only one sperm for the procedure to be successful. Even the conventional measures of sperm quality in terms of normal morphology or motility are not necessary for successful ICSI. Funding of andrological research has been neglected and improvement in assisted reproductive technology (ART) success has suffered as a consequence. Testing of sperm DNA damage shows much promise both as a diagnostic test for male infertility and a prognostic test for ART outcomes. It has been shown to be closely associated with numerous fertility outcomes including negative relationships with fertilization, embryo quality, implantation and positive relationships with miscarriage and childhood diseases. Here we report the relationships between in vitro fertilisation, ICSI pregnancy rates and sperm DNA damage, using the Comet assay to measure DNA fragmentation and also a novel test to measure modified bases, as a indication of oxidative DNA injury.

  11. 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

  12. Ghrelin Prevents Cisplatin-Induced Testicular Damage by Facilitating Repair of DNA Double Strand Breaks Through Activation of p53 in Mice.

    PubMed

    Garcia, Jose M; Chen, Ji-an; Guillory, Bobby; Donehower, Lawrence A; Smith, Roy G; Lamb, Dolores J

    2015-07-01

    Cisplatin administration induces DNA damage resulting in germ cell apoptosis and subsequent testicular atrophy. Although 50 percent of male cancer patients receiving cisplatin-based chemotherapy develop long-term secondary infertility, medical treatment to prevent spermatogenic failure after chemotherapy is not available. Under normal conditions, testicular p53 promotes cell cycle arrest, which allows time for DNA repair and reshuffling during meiosis. However, its role in the setting of cisplatin-induced infertility has not been studied. Ghrelin administration ameliorates the spermatogenic failure that follows cisplatin administration in mice, but the mechanisms mediating these effects have not been well established. The aim of the current study was to characterize the mechanisms of ghrelin and p53 action in the testis after cisplatin-induced testicular damage. Here we show that cisplatin induces germ cell damage through inhibition of p53-dependent DNA repair mechanisms involving gamma-H2AX and ataxia telangiectasia mutated protein kinase. As a result, testicular weight and sperm count and motility were decreased with an associated increase in sperm DNA damage. Ghrelin administration prevented these sequelae by restoring the normal expression of gamma-H2AX, ataxia telangiectasia mutated, and p53, which in turn allows repair of DNA double stranded breaks. In conclusion, these findings indicate that ghrelin has the potential to prevent or diminish infertility caused by cisplatin and other chemotherapeutic agents by restoring p53-dependent DNA repair mechanisms. PMID:26019260

  13. Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair

    PubMed Central

    Soares, Daniele G.; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M.; Henriques, João A. P.; Larsen, Annette K.; Escargueil, Alexandre E.

    2016-01-01

    Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors. PMID:27029031

  14. Mitochondrial DNA damage and efficiency of ATP biosynthesis: mathematical model.

    PubMed

    Beregovskaya, N; Maiboroda, R

    1995-01-21

    The role of mitochondrial DNA (mtDNA) damage in ageing processes and in malignant transformation of a cell is discussed. A mathematical model of the mtDNA population in a cell and in tissue is constructed. The model describes the effects of mtDNA damages accumulated during ageing and some features of malignant transformation and regeneration.

  15. DNA-damaging autoantibodies and cancer: the lupus butterfly theory.

    PubMed

    Noble, Philip W; Bernatsky, Sasha; Clarke, Ann E; Isenberg, David A; Ramsey-Goldman, Rosalind; Hansen, James E

    2016-07-01

    Autoantibodies reactive against host DNA are detectable in the circulation of most people with systemic lupus erythematosus (SLE). The long-held view that antibodies cannot penetrate live cells has been disproved. A subset of lupus autoantibodies penetrate cells, translocate to nuclei, and inhibit DNA repair or directly damages DNA. The result of these effects depends on the microenvironment and genetic traits of the cell. Some DNA-damaging antibodies alone have little impact on normal cells, but in the presence of other conditions, such as pre-existing DNA-repair defects, can become highly toxic. These findings raise new questions about autoimmunity and DNA damage, and reveal opportunities for new targeted therapies against malignancies particularly vulnerable to DNA damage. In this Perspectives article, we review the known associations between SLE, DNA damage and cancer, and propose a theory for the effects of DNA-damaging autoantibodies on SLE pathophysiology and cancer risk. PMID:27009542

  16. Initiation of DNA damage responses through XPG-related nucleases

    PubMed Central

    Kuntz, Karen; O'Connell, Matthew J

    2013-01-01

    Lesion-specific enzymes repair different forms of DNA damage, yet all lesions elicit the same checkpoint response. The common intermediate required to mount a checkpoint response is thought to be single-stranded DNA (ssDNA), coated by replication protein A (RPA) and containing a primer-template junction. To identify factors important for initiating the checkpoint response, we screened for genes that, when overexpressed, could amplify a checkpoint signal to a weak allele of chk1 in fission yeast. We identified Ast1, a novel member of the XPG-related family of endo/exonucleases. Ast1 promotes checkpoint activation caused by the absence of the other XPG-related nucleases, Exo1 and Rad2, the homologue of Fen1. Each nuclease is recruited to DSBs, and promotes the formation of ssDNA for checkpoint activation and recombinational repair. For Rad2 and Exo1, this is independent of their S-phase role in Okazaki fragment processing. This XPG-related pathway is distinct from MRN-dependent responses, and each enzyme is critical for damage resistance in MRN mutants. Thus, multiple nucleases collaborate to initiate DNA damage responses, highlighting the importance of these responses to cellular fitness. PMID:23211746

  17. Cu(II)-dipeptide complexes of 2-(4'-thiazolyl)benzimidazole: synthesis, DNA oxidative damage, antioxidant and in vitro antitumor activity.

    PubMed

    Fu, Xia-Bing; Zhang, Jia-Jia; Liu, Dan-Dan; Gan, Qian; Gao, Hong-Wei; Mao, Zong-Wan; Le, Xue-Yi

    2015-02-01

    Two new Cu(II)-dipeptide complexes of 2-(4'-thiazolyl)benzimidazole, [Cu(Gly-Gly)(TBZ)(Cl)]·4H2O (1) and [Cu(Gly-l-Leu)(TBZ)(Cl)]·H2O (2) (Gly-Gly=glycyl-glycine anion, Gly-l-Leu=glycyl-l-leucine anion and TBZ=2-(4'-thiazolyl)benzimidazole) have been synthesized and characterized by elemental analyses, molar conductance measurements and spectroscopy methods (IR, UV-visible, electrospray ionization mass spectra (ESI-MS) and EPR). The DNA binding and cleavage properties of the complexes monitored by multi-spectroscopic techniques (UV absorption, fluorescence and circular dichroism), viscosity determination and agarose gel electrophoresis indicated that the complexes bound to calf thymus (CT)-DNA via a partial intercalative mode with considerable intrinsic binding constants (Kb=1.64×10(5)M(-1) for 1 and 2.59×10(5)M(-1) for 2), and cleaved pBR322 DNA efficiently in the mediation of ascorbic acid (AA), probably via an oxidative damage mechanism induced by OH. The antioxidant activities of the complexes have been evaluated by means of modified nitroblue tetrazolium (NBT) photoreduction and cellular antioxidant activity (CAA) assays using HepG2 cells as a model, and it was found that IC50 values of 1 and 2 for dismutation of O2(-) were 0.172 and 0.247μM, respectively, and the CAA50 values were 10.57 and 10.74μM. In addition, the complexes were subjected to in vitro cytotoxicity against three human carcinoma cell lines (HeLa, A549 and HepG2), which revealed that the complexes exhibited effective cytotoxicity (IC50 values varying from 33.17 to 100μM) and selective inhibition toward HeLa cell lines. These findings indicate that the complexes have the potential to act as effective metallopeptide chemotherapeutic agents.

  18. Metallothionein blocks oxidative DNA damage in vitro

    PubMed Central

    Qu, Wei; Pi, Jingbo; Waalkes, Michael P.

    2012-01-01

    The role of metallothionein (MT) in mitigation of oxidative DNA damage (ODD) induced either by cadmium (Cd) or the direct oxidant hydrogen peroxide (H2O2) was systematically examined by using MT-I/II double knockout (MT-null) or MT-competent wild-type (WT) cells. Both toxicants were much more lethal to MT-null cells (Cd LC50 = 6.6 μM; H2O2 LC50 = 550 μM) than WT cells (Cd LC50 = 16.5 μM; H2O2 LC50 = 930 μM). Cd induced concentration-related MT increases in WT cells, while the basal levels were undetectable and not increased by Cd in MT-null cells. ODD, measured by the immuno-spin trapping method, was minimally induced by sub-toxic Cd levels (1 or 5 μM; 24 h) in WT cells, but markedly increased in MT-null cells (> 430%). Similarly, ODD was induced to higher levels by lower concentrations of H2O2 in MT-null cells than WT cells. Transfection of MT-I into MT-null cells reduced both Cd- and H2O2-induced cytolethality and ODD. Cd increased expression of the oxidant defense genes, HO-1 and GSTa2 to a much greater extent in MT-null cells than WT. Cd or H2O2 exposure increased expression of key transport genes, Mrp1 and Mrp2, in WT cells but not in MT-null cells. MT protects against Cd- and H2O2-induced ODD in MT competent cells possibly by multiple mechanisms, potentially including direct metal ion sequestration and sequestration of oxidant radicals by MT. MT-deficient cells appear to adapt to Cd primarily by turning on oxidant response systems, while MT-competent cells activate MT and transport systems. PMID:22914987

  19. Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

    PubMed

    Taylor, John-Stephen

    2015-12-01

    How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

  20. DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint

    PubMed Central

    Collins, Josie K.; Lane, Simon I. R.; Merriman, Julie A.; Jones, Keith T.

    2015-01-01

    Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis. PMID:26522232

  1. PNAS-4, an Early DNA Damage Response Gene, Induces S Phase Arrest and Apoptosis by Activating Checkpoint Kinases in Lung Cancer Cells*

    PubMed Central

    Yuan, Zhu; Guo, Wenhao; Yang, Jun; Li, Lei; Wang, Meiliang; Lei, Yi; Wan, Yang; Zhao, Xinyu; Luo, Na; Cheng, Ping; Liu, Xinyu; Nie, Chunlai; Peng, Yong; Tong, Aiping; Wei, Yuquan

    2015-01-01

    PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53−/−) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21Waf1/Cip1 and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21Waf1/Cip1 is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells. PMID:25918161

  2. Mechanism study of goldenseal-associated DNA damage.

    PubMed

    Chen, Si; Wan, Liqing; Couch, Letha; Lin, Haixia; Li, Yan; Dobrovolsky, Vasily N; Mei, Nan; Guo, Lei

    2013-07-31

    Goldenseal has been used for the treatment of a wide variety of ailments including gastrointestinal disturbances, urinary tract disorders, and inflammation. The five major alkaloid constituents in goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine. When goldenseal was evaluated by the National Toxicology Program (NTP) in the standard 2-year bioassay, goldenseal induced an increase in liver tumors in rats and mice; however, the mechanism of goldenseal-associated liver carcinogenicity remains unknown. In this study, the toxicity of the five goldenseal alkaloid constituents was characterized, and their toxic potencies were compared. As measured by the Comet assay and the expression of γ-H2A.X, berberine, followed by palmatine, appeared to be the most potent DNA damage inducer in human hepatoma HepG2 cells. Berberine and palmatine suppressed the activities of both topoisomerase (Topo) I and II. In berberine-treated cells, DNA damage was shown to be directly associated with the inhibitory effect of Topo II, but not Topo I by silencing gene of Topo I or Topo II. In addition, DNA damage was also observed when cells were treated with commercially available goldenseal extracts and the extent of DNA damage was positively correlated to the berberine content. Our findings suggest that the Topo II inhibitory effect may contribute to berberine- and goldenseal-induced genotoxicity and tumorigenicity.

  3. Ethanol-induced mitophagy in liver is associated with activation of the PINK1-Parkin pathway triggered by oxidative DNA damage.

    PubMed

    Eid, Nabil; Ito, Yuko; Horibe, Akio; Otsuki, Yoshinori

    2016-10-01

    Mitophagy is a cytoprotective mechanism against mitochondrial damaging agents. Studies demonstrating morphological evidence for the involvement of the PINK1-Parkin pathway in the hepatocyte mitophagic response to ethanol toxicity, and potential links to apoptosis and mitochondrial alterations such as spheroid formation are still lacking. We addressed these unresolved issues using a rat model of binge alcohol exposure. Adult rats were injected with ethanol (5g/kg) and liver samples were taken at 0, 3, 6, and 24 hours after ethanol administration and processed for light and electron microscopic studies. Ethanol induced a low level of hepatocyte apoptosis, peaking at 3 h and decreasing significantly by 24 h. In contrast, there was enhanced formation of mitophagic vacuoles in the majority of normal hepatocytes of ethanol-treated rats (ETRs), which peaked at 6 h and was maintained up to 24 h based on electron microscopy and TUNEL/LC3 double labelling. Moreover, enhanced mitophagy in ETR hepatocytes was confirmed by increased LC3 puncta formation, and co-localization of Parkin and LC3 with mitochondrial and lysosomal markers. Immunoelectron microscopy demonstrated the localization of PINK1 and Parkin to damaged mitochondria of ETR hepatocytes, which was consistent with co-localization of Parkin with 8-OHdG, a marker of oxidative mitochondrial DNA damage. Furthermore, electron microscopy showed enhanced formation of mitochondrial spheroids in ETR hepatocytes. These data are the first direct morphological evidence linking PINK1-Parkin pathway activation to the enhanced mitophagic response of hepatocytes to ethanol toxicity. Ethanol-induced hepatic mitophagy may be a prosurvival mechanism, which may have therapeutic implications. PMID:26935412

  4. 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

  5. Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

    PubMed

    Trabold, Peter A; Weinberger, Martin; Feng, Li; Burhans, William C

    2005-04-01

    Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA. PMID:15647270

  6. Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

    PubMed Central

    Schneider, L; Fumagalli, M; d'Adda di Fagagna, F

    2012-01-01

    The impact and consequences of damage generation into genomic DNA, especially in the form of DNA double-strand breaks, and of the DNA-damage response (DDR) pathways that are promptly activated, have been elucidated in great detail. Most of this research, however, has been performed on proliferating, often cancerous, cell lines. In a mammalian body, the majority of cells are terminally differentiated (TD), and derives from a small pool of self-renewing somatic stem cells. Here, we comparatively studied DDR signaling and radiosensitivity in neural stem cells (NSC) and their TD-descendants, astrocytes – the predominant cells in the mammalian brain. Astrocytes have important roles in brain physiology, development and plasticity. We discovered that NSC activate canonical DDR upon exposure to ionizing radiation. Strikingly, astrocytes proved radioresistant, lacked functional DDR signaling, with key DDR genes such as ATM being repressed at the transcriptional level. Nevertheless, astrocytes retain the expression of non-homologous end-joining (NHEJ) genes and indeed they are DNA repair proficient. Unlike in NSC, in astrocytes DNA-PK seems to be the PI3K-like protein kinase responsible for γH2AX signal generation upon DNA damage. We also demonstrate the lack of functional DDR signaling activation in vivo in astrocytes of irradiated adult mouse brains, although adjacent neurons activate the DDR. PMID:21979466

  7. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    PubMed Central

    Geng, Deyu; Zhang, Zhixia; Guo, Huarong

    2012-01-01

    p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. PMID:25585933

  8. Metal specificity in DNA damage prevention by sulfur antioxidants.

    PubMed

    Battin, Erin E; Brumaghim, Julia L

    2008-12-01

    Metals such as CuI and FeII generate hydroxyl radical (.OH) by reducing endogenous hydrogen peroxide (H2O2). Because antioxidants can ameliorate metal-mediated oxidative damage, we have quantified the ability of glutathione, a primary intracellular antioxidant, and other biological sulfur-containing compounds to inhibit metal-mediated DNA damage caused hydroxyl radical. In the CuI/H2O2 system, six sulfur compounds, including both reduced and oxidized glutathione, inhibited DNA damage with IC50 values ranging from 3.4 to 12.4 microM. Glutathione and 3-carboxypropyl disulfide also demonstrated significant antioxidant activity with FeII and H2O2. Additional gel electrophoresis and UV-vis spectroscopy studies confirm that antioxidant activity for sulfur compounds in the CuI system is attributed to metal coordination, a previously unexplored mechanism. The antioxidant mechanism for sulfur compounds in the FeII system, however, is unlike that of CuI. Our results demonstrate that glutathione and other sulfur compounds are potent antioxidants capable of preventing metal-mediated oxidative DNA damage at well below their biological concentrations. This novel metal-binding antioxidant mechanism may play a significant role in the antioxidant behavior of these sulfur compounds and help refine understanding of glutathione function in vivo. PMID:18675460

  9. Regulation of the DNA Damage Response by p53 Cofactors

    PubMed Central

    Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

    2012-01-01

    The selective expression of p53-targeted genes is central to the p53-mediated DNA damage response. It is affected by multiple factors including posttranslational modifications and cofactors of p53. Here, we proposed an integrated model of the p53 network to characterize how the cellular response is regulated by key cofactors of p53, Hzf and ASPP. We found that the sequential induction of Hzf and ASPP is crucial to a reliable cell-fate decision between survival and death. After DNA damage, activated p53 first induces Hzf, which promotes the expression of p21 to arrest the cell cycle and facilitate DNA repair. The cell recovers to normal proliferation after the damage is repaired. If the damage is beyond repair, Hzf is effectively degraded, and activated E2F1 induces ASPP, which promotes the expression of Bax to trigger apoptosis. Furthermore, interrupting the induction of Hzf or ASPP remarkably impairs the cellular function. We also proposed two schemes for the production of the unknown E3 ubiquitin ligase for Hzf degradation: it is induced by either E2F1 or p53. In both schemes, the sufficient degradation of Hzf is required for apoptosis induction. These results are in good agreement with experimental observations or are experimentally testable. PMID:22677378

  10. Uridine homeostatic disorder leads to DNA damage and tumorigenesis.

    PubMed

    Cao, Zhe; Ma, Jun; Chen, Xinchun; Zhou, Boping; Cai, Chuan; Huang, Dan; Zhang, Xuewen; Cao, Deliang

    2016-03-28

    Uridine is a natural nucleoside precursor of uridine monophosphate in organisms and thus is considered to be safe and is used in a wide range of clinical settings. The far-reaching effects of pharmacological uridine have long been neglected. Here, we report that the homeostatic disorder of uridine is carcinogenic. Targeted disruption (-/-) of murine uridine phosphorylase (UPase) disrupted the homeostasis of uridine and increased spontaneous tumorigenesis by more than 3-fold. Multiple tumors (e.g., lymphoma, hepatoma and lung adenoma) occurred simultaneously in some UPase deficient mice, but not in wild-type mice raised under the same conditions. In the tissue from UPase -/- mice, the 2'-deoxyuridine,5'-triphosphate (dUTP) levels and uracil DNA were increased and p53 was activated with an increased phospho-Ser18 p53 level. Exposing cell lines (e.g., MCF-7, RKO, HCT-8 and NCI-H460) to uridine (10 or 30 µM) led to uracil DNA damage and p53 activation, which in turn triggered the DNA damage response. In these cells, phospho-ATM, phospho-CHK2, and phospho-γH2AX were increased by uridine. These data suggest that uridine homeostatic disorder leads to uracil DNA damage and that pharmacological uridine may be carcinogenic.

  11. CC3/TIP30 affects DNA damage repair

    PubMed Central

    2010-01-01

    Background The pro-apoptotic protein CC3/TIP30 has an unusual cellular function as an inhibitor of nucleocytoplasmic transport. This function is likely to be activated under conditions of stress. A number of studies support the notion that CC3 acts as a tumor and metastasis suppressor in various types of cancer. The yeast homolog of CC3 is likely to be involved in responses to DNA damage. Here we examined the potential role of CC3 in regulation of cellular responses to genotoxic stress. Results We found that forced expression of CC3 in CC3-negative cells strongly delays the repair of UV-induced DNA damage. Exogenously introduced CC3 negatively affects expression levels of DDB2/XPE and p21CIP1, and inhibits induction of c-FOS after UV exposure. In addition, exogenous CC3 prevents the nuclear accumulation of P21CIP in response to UV. These changes in the levels/localization of relevant proteins resulting from the enforced expression of CC3 are likely to contribute to the observed delay in DNA damage repair. Silencing of CC3 in CC3-positive cells has a modest delaying effect on repair of the UV induced damage, but has a much more significant negative affect on the translesion DNA synthesis after UV exposure. This could be related to the higher expression levels and increased nuclear localization of p21CIP1 in cells where expression of CC3 is silenced. Expression of CC3 also inhibits repair of oxidative DNA damage and leads to a decrease in levels of nucleoredoxin, that could contribute to the reduced viability of CC3 expressing cells after oxidative insult. Conclusions Manipulation of the cellular levels of CC3 alters expression levels and/or subcellular localization of proteins that exhibit nucleocytoplasmic shuttling. This results in altered responses to genotoxic stress and adversely affects DNA damage repair by affecting the recruitment of adequate amounts of required proteins to proper cellular compartments. Excess of cellular CC3 has a significant negative

  12. Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions

    PubMed Central

    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

  13. Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange.

    PubMed

    Oliver, Antony W; Paul, Angela; Boxall, Katherine J; Barrie, S Elaine; Aherne, G Wynne; Garrett, Michelle D; Mittnacht, Sibylle; Pearl, Laurence H

    2006-07-12

    The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.

  14. Changing the ubiquitin landscape during viral manipulation of the DNA damage response

    PubMed Central

    Weitzman, Matthew D.; Lilley, Caroline E.; Chaurushiya, Mira S.

    2011-01-01

    Viruses often induce signaling through the same cellular cascades that are activated by damage to the cellular genome. Signaling triggered by viral proteins or exogenous DNA delivered by viruses can be beneficial or detrimental to viral infection. Viruses have therefore evolved to dissect the cellular DNA damage response pathway during infection, often marking key cellular regulators with ubiquitin to induce their degradation or change their function. Signaling controlled by ubiquitin or ubiquitin-like proteins has recently emerged as key regulator of the cellular DNA damage response. Situated at the interface between DNA damage signaling and the ubiquitin system, viruses can reveal key convergence points in this important cellular pathway. In this review, we examine how viruses harness the diversity of the cellular ubiquitin system to modulate the DNA damage signaling pathway. We discuss the implications of viral infiltration of this pathway for both the transcriptional program of the virus and for the cellular response to DNA damage. PMID:21549706

  15. Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes.

    PubMed

    Lee, Kyungeun; Kim, Deok Ryong; Ahn, Byungchan

    2004-08-31

    The DNA repair machinery must locate and repair DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo. To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment. Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities. PMID:15359130

  16. Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

    SciTech Connect

    Nagane, Masaki; Yasui, Hironobu; Sakai, Yuri; Yamamori, Tohru; Niwa, Koichi; Hattori, Yuichi; Kondo, Takashi; Inanami, Osamu

    2015-01-02

    Highlights: • eNOS activity is increased in BAECs exposed to X-rays. • ATM is involved in this increased eNOS activity. • HSP90 modulates the radiation-induced activation of ATM and eNOS. - Abstract: In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90.

  17. Role of DNA damage in atherosclerosis--bystander or participant?

    PubMed

    Gray, Kelly; Bennett, Martin

    2011-10-01

    Atherosclerosis leading to cardiovascular disease is the leading cause of death among western populations. Atherosclerosis in characterised by the development of a fibrofatty lesion that consists of a diverse cell population, including inflammatory cells that create an intensely oxidising environment within the vessel. Coupled with normal replication, the local intracellular and extracellular environment causes damage to cellular DNA that is recognised and repaired by the DNA damage response (DDR) pathway. The role of DNA damage and the resulting deregulation of 'normal' cellular behaviour and subsequent loss of cell cycle control checkpoints have been widely studied in cancer. However, despite the extensive evidence for DNA damage in atherosclerosis, it is only over the past two decades that a causative link between DNA damage and atherosclerosis has been hypothesised. Whilst atherosclerosis is a feature of human disease characterised by defects in DNA damage, currently the role of DNA damage in the initiation and progression of atherosclerosis remains highly debated, as a 'chicken and egg' situation. This review will analyse the evidence for, the causes of, and consequences of DNA damage in atherosclerosis, detail the DNA damage response pathway that results in these consequences, and highlight therapeutic opportunities in this area. We also outline the evidence that DNA damage is a cause of both initiation and progression of atherosclerosis, and not just a consequence of disease.

  18. 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

  19. p53 in the DNA-Damage-Repair Process.

    PubMed

    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 providing protection from cancer development by maintaining genome stability.

  20. p53 in the DNA-Damage-Repair Process.

    PubMed

    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 providing protection from cancer development by maintaining genome stability. PMID:27048304

  1. 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

  2. Defects in mitochondrial DNA replication and oxidative damage in muscle of mtDNA mutator mice.

    PubMed

    Kolesar, Jill E; Safdar, Adeel; Abadi, Arkan; MacNeil, Lauren G; Crane, Justin D; Tarnopolsky, Mark A; Kaufman, Brett A

    2014-10-01

    A causal role for mitochondrial dysfunction in mammalian aging is supported by recent studies of the mtDNA mutator mouse ("PolG" mouse), which harbors a defect in the proofreading-exonuclease activity of mitochondrial DNA polymerase gamma. These mice exhibit accelerated aging phenotypes characteristic of human aging, including systemic mitochondrial dysfunction, exercise intolerance, alopecia and graying of hair, curvature of the spine, and premature mortality. While mitochondrial dysfunction has been shown to cause increased oxidative stress in many systems, several groups have suggested that PolG mutator mice show no markers of oxidative damage. These mice have been presented as proof that mitochondrial dysfunction is sufficient to accelerate aging without oxidative stress. In this study, by normalizing to mitochondrial content in enriched fractions we detected increased oxidative modification of protein and DNA in PolG skeletal muscle mitochondria. We separately developed novel methods that allow simultaneous direct measurement of mtDNA replication defects and oxidative damage. Using this approach, we find evidence that suggests PolG muscle mtDNA is indeed oxidatively damaged. We also observed a significant decrease in antioxidants and expression of mitochondrial biogenesis pathway components and DNA repair enzymes in these mice, indicating an association of maladaptive gene expression with the phenotypes observed in PolG mice. Together, these findings demonstrate the presence of oxidative damage associated with the premature aging-like phenotypes induced by mitochondrial dysfunction.

  3. 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.

  4. Nile blue can photosensitize DNA damage through electron transfer.

    PubMed

    Hirakawa, Kazutaka; Ota, Kazuhiro; Hirayama, Junya; Oikawa, Shinji; Kawanishi, Shosuke

    2014-04-21

    The mechanism of DNA damage photosensitized by Nile blue (NB) was studied using (32)P-5'-end-labeled DNA fragments. NB bound to the DNA strand was possibly intercalated through an electrostatic interaction. Photoirradiated NB caused DNA cleavage at guanine residues when the DNA fragments were treated with piperidine. Consecutive guanines, the underlined G in 5'-GG and 5'-GGG, were selectively damaged through photoinduced electron transfer. The fluorescence lifetime of NB was decreased by guanine-containing DNA sequence, supporting this mechanism. Single guanines were also slightly damaged by photoexcited NB, and DNA photodamage by NB was slightly enhanced in D2O. These results suggest that the singlet oxygen mechanism also partly contributes to DNA photodamage by NB. DNA damage photosensitized by NB via electron transfer may be an important mechanism in medicinal applications of photosensitizers, such as photodynamic therapy in low oxygen. PMID:24576317

  5. Nitroxides block DNA scission and protect cells from oxidative damage

    SciTech Connect

    Samuni, A.; Godinger, D.; Aronovitch, J. ); Russo, A.; Mitchell, J. )

    1991-01-01

    The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H{sub 2}O{sub 2}. Oxidative damage induced by H{sub 2}O{sub 2} was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated with H{sub 2}O{sub 2}-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H{sub 2}O{sub 2} cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H{sub 2}O{sub 2} toxicity, without lowering H{sub 2}O{sub 2} concentration. To check whether nitroxides protect against O{sub 2}{sup {center dot}{minus}}-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H{sub 2}O{sub 2}, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.

  6. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

    PubMed Central

    Nacerddine, Karim; Beaudry, Jean-Bernard; Ginjala, Vasudeva; Westerman, Bart; Mattiroli, Francesca; Song, Ji-Ying; van der Poel, Henk; Ponz, Olga Balagué; Pritchard, Colin; Cornelissen-Steijger, Paulien; Zevenhoven, John; Tanger, Ellen; Sixma, Titia K.; Ganesan, Shridar; van Lohuizen, Maarten

    2012-01-01

    Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis. PMID:22505453

  7. 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.

  8. rasiRNAs, DNA damage, and embryonic axis specification.

    PubMed

    Theurkauf, W E; Klattenhoff, C; Bratu, D P; McGinnis-Schultz, N; Koppetsch, B S; Cook, H A

    2006-01-01

    Drosophila repeat-associated small interfering RNAs (rasiRNAs) have been implicated in retrotransposon and stellate locus silencing. However, mutations in the rasiRNA pathway genes armitage, spindle-E, and aubergine disrupt embryonic axis specification, triggering defects in microtubule organization and localization of osk and grk mRNAs during oogenesis. We show that mutations in mei-41 and mnk, which encode ATR and Chk2 kinases that function in DNA damage signal transduction, dramatically suppress the cytoskeletal and RNA localization defects associated with rasiRNA mutations. In contrast, stellate and retrotransposon silencing are not restored in mei-41 and mnk double mutants. We also find that armitage, aubergine, and spindle-E mutations lead to germ-line-specific accumulation of gamma-H2Av foci, which form at DNA double-strand breaks, and that mutations in armi lead to Chk2-dependent phosphorylation of Vasa, an RNA helicase required for axis specification. The Drosophila rasiRNA pathway thus appears to suppress DNA damage in the germ line, and mutations in this pathway block axis specification by activating an ATR/Chk2-dependent DNA damage response that disrupts microtubule polarization and RNA localization.

  9. 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

  10. Decreased paraoxonase1 activity and increased malondialdehyde and oxidative DNA damage levels in primary open angle glaucoma

    PubMed Central

    Mumcu, Ugur Yilmaz; Kocer, Ibrahim; Ates, Orhan; Alp, H. Hakan

    2016-01-01

    To investigate the malondialdehyde (MDA) levels, paraoxonase1 (PON1) activity and 8-hydroxy 2-deoxyguanosine (8-OHdG) levels in the primary open angle glaucoma (POAG) patient. Blood samples from 52 healthy individuals and 53 patients with POAG were analyzed for MDA and 8-OHdG by HPLC (high-performance liquid chromatography) and PON1 by spectrophotometry. The data obtained were analyzed statistically. MDA levels were 10.46±8.4 and 4.70±1.79 µmol; PON1 levels were 121±39.55 and 161.62±60.22 U/mL; and 8-OHdG values were 1.32±0.53/106 dG and 0.47±0.27/106 dG in the POAG patients and the control group, respectively. The difference was significant in MDA levels, 8-OHdG levels and PON1 activity in POAG patients in comparison with controls (P<0.001). We concluded that the observed increase in MDA and 8-OHdG levels may be correlated with decreased PON1 activity. Oxidative stress plays an important role in glaucoma development. PMID:27803873

  11. Clustered DNA damage induced by heavy ion particles.

    PubMed

    Terato, Hiroaki; Ide, Hiroshi

    2004-12-01

    Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage. PMID:15858387

  12. 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.

  13. Molecular Analysis of a Multistep Lung Cancer Model Induced by Chronic Inflammation Reveals Epigenetic Regulation of p16 and Activation of the DNA Damage Response Pathway12

    PubMed Central

    Blanco, David; Vicent, Silvestre; Fraga, Mario F; Fernandez-Garcia, Ignacio; Freire, Javier; Lujambio, Amaia; Esteller, Manel; Ortiz-de-Solorzano, Carlos; Pio, Ruben; Lecanda, Fernando; Montuenga, Luis M

    2007-01-01

    The molecular hallmarks of inflammation-mediated lung carcinogenesis have not been fully clarified, mainly due to the scarcity of appropriate animal models. We have used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers and genetic alterations. We analyzed markers of DNA damage response (DDR), proliferative stress, and telomeric stress: γ-H2AX, p16, p53, and TERT. Lung cancer-related epigenetic and genetic alterations, including promoter hypermethylation status of p16(CDKN2A), APC, CDH13, Rassf1, and Nore1A, as well as mutations of Tp53, epidermal growth factor receptor, K-ras, N-ras, and c-H-ras, have been also studied. Our results showed DDR pathway activation in preneoplastic lesions, in association with inducible nitric oxide synthase and p53 induction. p16 was also induced in early tumorigenic progression and was inactivated in bronchiolar dysplasias and tumors. Remarkably, lack of mutations of Ras and epidermal growth factor receptor, and a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16(CDKN2A), CDH13, and APC, but not in Rassf1 and Nore1A, were clearly observed. These data suggest the existence of a specific molecular signature of inflammation-driven lung carcinogenesis that shares some, but not all, of the molecular landmarks of chemically induced lung cancer. PMID:17971904

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

    SciTech Connect

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

    2013-06-01

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

  15. 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

  16. DNA damage and L1 retrotransposition.

    PubMed

    Farkash, Evan A; Luning Prak, Eline T

    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

  17. DNA damage and L1 retrotransposition.

    PubMed

    Farkash, Evan A; Luning Prak, Eline T

    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.

  18. Lycopene-induced hydroxyl radical causes oxidative DNA damage in Escherichia coli.

    PubMed

    Lee, Wonyoung; Lee, Dong Gun

    2014-09-01

    Lycopene, which is a well-known red carotenoid pigment, has been drawing scientific interest because of its potential biological functions. The current study reports that lycopene acts as a bactericidal agent by inducing reactive oxygen species (ROS)-mediated DNA damage in Escherichia coli. Lycopene treatment elevated the level of ROS-in particular, hydroxyl radicals ((•)OH) -which can damage DNA in E. coli. Lycopene-induced DNA damage in bacteria was confirmed and we also observed cell filamentation caused by cell division arrest, an indirect marker of the DNA damage repair system, in lycopene-treated E. coli. Increased RecA expression was observed, indicating activation of the DNA repair system (SOS response). To summarize, lycopene exerts its antibacterial effects by inducing (•)OH -mediated DNA damage that cannot be ameliorated by the SOS response. Lycopene may be a clinically useful adjuvant for current antimicrobial therapies.

  19. DNA damage by smoke: Protection by turmeric and other inhibitors of ROS

    SciTech Connect

    Srinivas, L.; Shalini, V.K. )

    1991-01-01

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

  20. 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.

  1. The DNA Damage Response: Making it safe to play with knives

    PubMed Central

    Ciccia, Alberto; Elledge, Stephen J.

    2010-01-01

    Damage to our genetic material is an ongoing threat to both our ability to faithfully transmit genetic information to our offspring as well as our own survival. To respond to these threats, eukaryotes have evolved the DNA Damage Response (DDR). The DDR is a complex signal transduction pathway that has the ability to sense DNA damage and transduce this information to the cell to influence cellular responses to DNA damage. Cells possess an arsenal of enzymatic tools capable of remodeling and repairing DNA, however, their activities must be tightly regulated in a temporal, spatial and DNA lesion-appropriate fashion to optimize repair and prevent unnecessary and potentially deleterious alterations in the structure of DNA during normal cellular processes. This review will focus on how the DDR controls DNA repair and the phenotypic consequences of defects in these critical regulatory functions in mammals. PMID:20965415

  2. DNA Damage Response and Tumorigenesis in Mcm2 Deficient Mice

    PubMed Central

    Kunnev, Dimiter; Rusiniak, Michael E.; Kudla, Angela; Freeland, Amy; Cady, Gillian K.; Pruitt, Steven C.

    2010-01-01

    Mini-chromosome maintenance proteins (Mcm’s) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcm proteins has been shown to result in highly penetrant early onset cancers (Shima et al., 2007; Pruitt et al., 2007 and stem cell deficiencies (Pruitt et al., 2007). Here we use MEFs from an Mcm2 deficient strain of mice to show by DNA fiber analysis that origin usage is decreased in Mcm2 deficient cells under conditions of HU mediated replication stress. DNA damage responses (DDR) resulting from HU and additional replication dependent and independent genotoxic agents were also examined and shown to function at wild type levels. Further, basal levels of many components of the DNA damage response were expressed at wild type levels demonstrating that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5–2 fold increases in the basal levels of γ-H2AX, p21cip1 and 53bp foci were found, consistent with a slight chronic elevation in DDR pathways. The one condition in which a larger difference between wt and Mcm2 deficient cells was found occurred following UV irradiation and may reflect the role of Chk1 mediated suppression of dormant origins. In vivo, abrogating p53 mediated DDR in Mcm2 deficient mice results in increased embryonic lethality and accelerated cancer formation in surviving mice. Further, p53 mutation rescues the negative effect of Mcm2 deficiency on the survival of neural stem cells in vitro; however, the enhanced survival correlates with increased genetic damage relative to Mcm2 wt cells carrying the p53 mutation. Together these results demonstrate that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. Additionally, these studies demonstrate that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53. PMID:20440269

  3. ATP-dependent chromatin remodeling in the DNA-damage response

    PubMed Central

    2012-01-01

    The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways. PMID:22289628

  4. Oxidative DNA damage stalls the human mitochondrial replisome

    PubMed Central

    Stojkovič, Gorazd; Makarova, Alena V.; Wanrooij, Paulina H.; Forslund, Josefin; Burgers, Peter M.; Wanrooij, Sjoerd

    2016-01-01

    Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism. PMID:27364318

  5. 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

  6. 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.

  7. DNA damage and neurotoxicity of chronic alcohol abuse

    PubMed Central

    Kruman, Inna I; Henderson, George I; Bergeson, Susan E

    2013-01-01

    Chronic alcohol abuse results in a variety of pathological effects including damage to the brain. The causes of alcohol-induced brain pathology are presently unclear. Several mechanisms of pathogenicity of chronic alcoholism have been proposed, including accumulation of DNA damage in the absence of repair, resulting in genomic instability and death of neurons. Genomic instability is a unified genetic mechanism leading to a variety of neurodegenerative disorders. Ethanol also likely interacts with various metabolic pathways, including one-carbon metabolism (OCM). OCM is critical for the synthesis of DNA precursors, essential for DNA repair, and as a methyl donor for various methylation events, including DNA methylation. Both DNA repair and DNA methylation are critical for maintaining genomic stability. In this review, we outline the role of DNA damage and DNA repair dysfunction in chronic alcohol-induced neurodegeneration. PMID:22829701

  8. 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

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

    PubMed

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

    2014-12-30

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

  10. 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.

  11. DNA Damage among Wood Workers Assessed with the Comet Assay.

    PubMed

    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.

  12. 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

  13. DNA Damage among Wood Workers Assessed with the Comet Assay.

    PubMed

    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

  14. Purine Analog-Like Properties of Bendamustine Underlie Rapid Activation of DNA Damage Response and Synergistic Effects with Pyrimidine Analogues in Lymphoid Malignancies

    PubMed Central

    Hiraoka, Nobuya; Kikuchi, Jiro; Yamauchi, Takahiro; Koyama, Daisuke; Wada, Taeko; Uesawa, Mitsuyo; Akutsu, Miyuki; Mori, Shigehisa; Nakamura, Yuichi; Ueda, Takanori; Kano, Yasuhiko; Furukawa, Yusuke

    2014-01-01

    Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies. PMID:24626203

  15. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma

    PubMed Central

    Cottini, Francesca; Hideshima, Teru; Suzuki, Rikio; Tai, Yu-Tzu; Bianchini, Giampaolo; Richardson, Paul G.; Anderson, Kenneth C.; Tonon, Giovanni

    2015-01-01

    Ongoing DNA damage is a common feature of epithelial cancers. Here we show that tumor cells derived from multiple myeloma (MM), a disease of clonal plasma cells, demonstrate DNA replicative stress leading to DNA damage. We identified a poor prognosis subset of MM with extensive chromosomal instability and replicative stress which rely on ATR to compensate for DNA replicative stress; conversely, silencing of ATR or treatment with a specific ATR inhibitor triggers MM cell apoptosis. We show that oncogenes such as MYC induce DNA damage in MM cells not only by increased replicative stress, but also via increased oxidative stress, and that ROS-inducer piperlongumine triggers further DNA damage and apoptosis. Importantly, ATR inhibition combined with piperlongumine triggers synergistic MM cytotoxicity. This synthetic lethal approach, enhancing oxidative stress while concomitantly blocking replicative stress response, provides a novel combination targeted therapy to address an unmet medical need in this subset of MM. PMID:26080835

  16. Evaluation of cytotoxicity and DNA damage response with analysis of intracellular ATM signaling pathways.

    PubMed

    Bandi, Sriram; Viswanathan, Preeti; Gupta, Sanjeev

    2014-06-01

    Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs. PMID:24927134

  17. Nuclear DNA damage as a direct cause of aging.

    PubMed

    Best, Benjamin P

    2009-06-01

    This paper presents evidence that damage to nuclear DNA (nDNA) is a direct cause of aging in addition to the effects of nDNA damage on cancer, apoptosis, and cellular senescence. Many studies show significant nDNA damage with age, associated with declining nDNA repair, and this evidence for the decline of nDNA repair with age is also reviewed. Mammalian lifespans correlate with the effectiveness of nDNA repair. The most severe forms of accelerated aging disease in humans are due to nDNA repair defects, and many of these diseases do not exhibit increased cancer incidence. High rates of cellular senescence and apoptosis due to high rates of nDNA damage are apparently the main cause of the elderly phenotype in these diseases. Transgenic mice with high rates of cellular senescence and apoptosis exhibit an elderly phenotype, whereas some strains with low rates of cellular senescence and apoptosis show extended lifespan. Age-associated increases of nDNA damage in the brain may be problematic for rejuvenation because neurons may be difficult to replace and artificial nDNA repair could be difficult. PMID:19594328

  18. 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

  19. Nuclear DNA damage as a direct cause of aging.

    PubMed

    Best, Benjamin P

    2009-06-01

    This paper presents evidence that damage to nuclear DNA (nDNA) is a direct cause of aging in addition to the effects of nDNA damage on cancer, apoptosis, and cellular senescence. Many studies show significant nDNA damage with age, associated with declining nDNA repair, and this evidence for the decline of nDNA repair with age is also reviewed. Mammalian lifespans correlate with the effectiveness of nDNA repair. The most severe forms of accelerated aging disease in humans are due to nDNA repair defects, and many of these diseases do not exhibit increased cancer incidence. High rates of cellular senescence and apoptosis due to high rates of nDNA damage are apparently the main cause of the elderly phenotype in these diseases. Transgenic mice with high rates of cellular senescence and apoptosis exhibit an elderly phenotype, whereas some strains with low rates of cellular senescence and apoptosis show extended lifespan. Age-associated increases of nDNA damage in the brain may be problematic for rejuvenation because neurons may be difficult to replace and artificial nDNA repair could be difficult.

  20. 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. PMID:26082923

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

    NASA Astrophysics Data System (ADS)

    Ramesh, Govindarajan; Wu, Honglu

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

  2. DNA damage response during mouse oocyte maturation.

    PubMed

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

    2016-01-01

    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

  3. Molecular Pathways: Overcoming Radiation Resistance by Targeting DNA Damage Response Pathways

    PubMed Central

    Morgan, Meredith A.; Lawrence, Theodore S.

    2015-01-01

    DNA double-strand breaks are the critical lesions responsible for the majority of ionizing radiation-induced cell killing. Thus, the ability of tumor cells to elicit a DNA damage response following radiation, via activation of DNA repair and cell cycle checkpoints, promotes radiation resistance and tumor cell survival. Consequently, agents which target these DNA damage response pathways are being developed to overcome radiation resistance. Overall, these agents are effective radiosensitizers; however, their mechanisms of tumor cell selectivity are not fully elucidated. In this review, we will focus on the crucial radiation-induced DNA damage responses as well as clinical and translational advances with agents designed to inhibit these responses. Importantly, we describe how synthetic lethality can provide tumor cell selective radiosensitization by these agents and expand the therapeutic window for DNA damage response-targeted agents used in combination with radiation therapy. PMID:26133775

  4. 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

  5. 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.

  6. Metabolic activation of tris(2,3-dibromopropyl)phosphate to reactive intermediates. II. Covalent binding, reactive metabolite formation, and differential metabolite-specific DNA damage in vivo.

    PubMed

    Pearson, P G; Omichinski, J G; Holme, J A; McClanahan, R H; Brunborg, G; Søderlund, E J; Dybing, E; Nelson, S D

    1993-02-01

    Analogs of tris(2,3-dibromopropyl)phosphate (Tris-BP) either labeled at specific positions with carbon-14 and phosphorus-32 or dual-labeled with both deuterium and tritium were administered to male Wistar rats at a nephrotoxic dose of 360 mumol/kg. The covalent binding of Tris-BP metabolites to hepatic, renal, and testicular proteins was determined after 9 and 24 hr, and plasma concentrations of bis(2,3-dibromopropyl)-phosphate (Bis-BP) formed metabolically from Tris-BP were measured at intervals throughout the initial 9-hr postdosing period. The covalent binding of 14C-Tris-BP metabolites in the kidney (2495 +/- 404 pmol/mg protein) was greater than that in the liver (476 +/- 123 pmol/mg protein) or testes (94 +/- 11 pmol/mg protein); the extent of renal covalent protein binding of Tris-BP metabolites was decreased by 82 and 84% when deuterium was substituted at carbon-2 and carbon-3, respectively. Substitution of Tris-BP with deuterium at carbon-2 or carbon-3 also decreased the mean area under the curve for Bis-BP plasma concentration by 48 and 57%, respectively. The mechanism of Tris-BP-induced renal and hepatic DNA damage was evaluated in Wistar rats by an automated alkaline elution procedure after the administration of analogs of Tris-BP or Bis-BP labeled at specific positions with deuterium. Renal DNA damage was decreased when Tris-BP was substituted with deuterium at either carbon-2 or carbon-3; the magnitude of the change correlated with both a decrease in the area under the Bis-BP plasma curve and a decrease in renal covalent binding of Tris-BP metabolites for each of the deuterated analogs. In marked contrast, analogs of Bis-BP labeled with deuterium at carbon-2 or carbon-3 did not show a decrease in the severity of renal DNA damage compared to unlabeled Bis-BP. On the basis of these observations a metabolic scheme for hepatic P-450-mediated oxidation at either carbon-2 or carbon-3 of Tris-BP affording Bis-BP by two alternate pathways that are susceptible

  7. Homogenous repair of singlet oxygen-induced DNA damage in differentially transcribed regions and strands of human mitochondrial DNA.

    PubMed Central

    Anson, R M; Croteau, D L; Stierum, R H; Filburn, C; Parsell, R; Bohr, V A

    1998-01-01

    Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) of human fibroblasts in culture. The primary product of this reaction is the DNA lesion 7-hydro-8-oxo-deoxyguanosine (8-oxo-dG). The DNA damage was quantitated using Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) in a gene-specific damage and repair assay. Assay conditions were refined to give incision at all enzyme-sensitive sites with minimal non-specific cutting. Cultured fibroblasts were exposed to photoactivated methylene blue under conditions that would produce an average of three oxidative lesions per double-stranded mitochondrial genome. Within 9 h, 47% of this damage had been removed by the cells. This removal was due to repair rather than to replication, cell loss or degradation of damaged genomes. The rate of repair was measured in both DNA strands of the frequently transcribed ribosomal region of the mitochondrial genome and in both strands of the non-ribosomal region. Fpg-sensitive alkali-resistant oxidative base damage was efficiently removed from human mtDNA with no differences in the rate of repair between strands or between two different regions of the genome that differ substantially with regard to transcriptional activity. PMID:9421531

  8. The yeast copper response is regulated by DNA damage.

    PubMed

    Dong, Kangzhen; Addinall, Stephen G; Lydall, David; Rutherford, Julian C

    2013-10-01

    Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast.

  9. The Yeast Copper Response Is Regulated by DNA Damage

    PubMed Central

    Dong, Kangzhen; Addinall, Stephen G.; Lydall, David

    2013-01-01

    Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast. PMID:23959798

  10. 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...

  11. Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage, antiproliferation and apoptotic induction activity in human cervical cancer cells.

    PubMed

    Qiao, Xin; Ma, Zhong-Ying; Shao, Jia; Bao, Wei-Guo; Xu, Jing-Yuan; Qiang, Zhao-Yan; Lou, Jian-Shi

    2014-02-01

    Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl2]·(CH3OH) (1) [BMA = N,N'-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K b = 1.24 × 10(4 )M(-1)) and the apparent binding constant (K app = 6.67 × 10(6 )M(-1)) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.

  12. Capturing snapshots of APE1 processing DNA damage.

    PubMed

    Freudenthal, Bret D; Beard, William A; Cuneo, Matthew J; Dyrkheeva, Nadezhda S; Wilson, Samuel H

    2015-11-01

    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. 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. 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. These snapshots provide mechanistic clarity for APE1 while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

  13. Capturing snapshots of APE1 processing DNA damage

    DOE PAGES

    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 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

  14. 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.

  15. 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

  16. 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.

  17. Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2

    PubMed Central

    Grindel, Annemarie; Guggenberger, Bianca; Eichberger, Lukas; Pöppelmeyer, Christina; Gschaider, Michaela; Tosevska, Anela; Mare, George; Briskey, David; Brath, Helmut; Wagner, Karl-Heinz

    2016-01-01

    Background Diabetes mellitus type 2 (T2DM) is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration. Methods Female T2DM patients (n = 146) were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c) level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72). In addition, tertiles according to diabetes duration (DD) were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49). Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals. Results No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group. Conclusion BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which

  18. AID and APOBEC deaminases: balancing DNA damage in epigenetics and immunity.

    PubMed

    Franchini, Don-Marc; Petersen-Mahrt, Svend K

    2014-01-01

    DNA mutations and genomic recombinations are the origin of oncogenesis, yet parts of developmental programs as well as immunity are intimately linked to, or even depend on, such DNA damages. Therefore, the balance between deleterious DNA damages and organismal survival utilizing DNA editing (modification and repair) is in continuous flux. The cytosine deaminases AID/APOBEC are a DNA editing family and actively participate in various biological processes. In conjunction with altered DNA repair, the mutagenic potential of the family allows for APOBEC3 proteins to restrict viral infection and transposons propagation, while AID can induce somatic hypermutation and class switch recombination in antibody genes. On the other hand, the synergy between effective DNA repair and the nonmutagenic potential of the DNA deaminases can induce local DNA demethylation to support epigenetic cellular identity. Here, we review the current state of knowledge on the mechanisms of action of the AID/APOBEC family in immunity and epigenetics.

  19. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells.

    PubMed

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells' molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  20. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  1. GSE4, a Small Dyskerin- and GSE24.2-Related Peptide, Induces Telomerase Activity, Cell Proliferation and Reduces DNA Damage, Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells

    PubMed Central

    Iarriccio, Laura; Manguán-García, Cristina; Pintado-Berninches, Laura; Mancheño, José Miguel; Molina, Antonio; Perona, Rosario; Sastre, Leandro

    2015-01-01

    Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients. PMID:26571381

  2. GSE4, a Small Dyskerin- and GSE24.2-Related Peptide, Induces Telomerase Activity, Cell Proliferation and Reduces DNA Damage, Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells.

    PubMed

    Iarriccio, Laura; Manguán-García, Cristina; Pintado-Berninches, Laura; Mancheño, José Miguel; Molina, Antonio; Perona, Rosario; Sastre, Leandro

    2015-01-01

    Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.

  3. GSE4, a Small Dyskerin- and GSE24.2-Related Peptide, Induces Telomerase Activity, Cell Proliferation and Reduces DNA Damage, Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells.

    PubMed

    Iarriccio, Laura; Manguán-García, Cristina; Pintado-Berninches, Laura; Mancheño, José Miguel; Molina, Antonio; Perona, Rosario; Sastre, Leandro

    2015-01-01

    Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients. PMID:26571381

  4. DNA damage control: regulation and functions of checkpoint kinase 1.

    PubMed

    Smits, Veronique A J; Gillespie, David A

    2015-10-01

    Checkpoint kinase 1 (Chk1) is a master regulator of the DNA damage and replication checkpoints in vertebrate cells. When activated via phosphorylation by its upstream regulatory kinase, ATR, Chk1 prevents cells with damaged or incompletely replicated DNA from entering mitosis, and acts to stabilize stalled replication forks and suppress replication origin firing when DNA synthesis is inhibited. Chk1 blocks mitosis by maintaining high levels of inhibitory tyrosine phosphorylation of the mitotic cyclin-dependent kinase 1; however, the mechanisms that underlie replication fork stabilization and suppression of origin firing are less well defined. Although Chk1 function is evidently acutely regulated during these responses, how this occurs at the molecular level is incompletely understood. Recent evidence that Chk1 contains a 'kinase-associated 1' domain within its regulatory C-terminal region promises new insights. Additional modifications catalysed by other protein kinases, such as cyclin-dependent kinase 1, Akt, and RSK, can combine with ubiquitylation to regulate Chk1 subcellular localization and protein stability. Interestingly, it is clear that Chk1 has less well-defined functions in homologous recombination, chromatin modification, gene expression, spindle checkpoint proficiency, and cytokinesis. Here, we provide an overview of Chk1 regulation and functions, with an emphasis on unresolved questions that merit further research. PMID:26216057

  5. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    PubMed

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress. PMID:27407148

  6. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

    PubMed Central

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F.; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-01-01

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker–induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress. PMID:27407148

  7. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    PubMed

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

  8. 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.

  9. DNA damage promotes Herpes Simplex Virus-1 protein expression in a neuroblastoma cell line

    PubMed Central

    Volcy, Ketna; Fraser, Nigel W.

    2013-01-01

    Although the induction of the cellular DNA damage response by Herpes simplex virus-1 (HSV-1) infection of epithelial cells in tissue culture promotes productive infection, there has been no experimental observation of the effect of the cellular DNA damage response on HSV-1 infection in vivo or in neuronal derived cell lines in tissue culture. Thus, it has been speculated that the lack of cellular DNA damage induction during infection of neurons may promote latency in these cells. This work examines the profile of HSV-1 promoter induction and protein expression, in the absence or presence of infection; using cellular DNA damage inducing topoisomerase inhibitors (Camptothecin and Etoposide) on a neuroblastoma cell line (C1300) in which HSV-1 infection fails to induce the DNA damage response. In the absence of infection, a plasmid expressing the immediate early ICP0 promoter was the most induced by the DNA damage drug treatments compared to the early (RR) and late (VP16) gene promoters. Similarly, drug treatment of C1300 cells infected with HSV-1 virus showed enhanced protein expression for ICP0, but not ICP4 and VP16 proteins. However, when the cells were infected with a HSV-1 virus defective in the immediate early gene trans-activator VP16 (in814) and treated with the DNA damaging drugs, there was enhanced expression of immediate early and late HSV-1 proteins. Although, viral infection of the neuroblastoma cell alone did not induce DNA damage, cellular DNA damage induced by drug treatments facilitated viral promoter induction and viral protein expression. This implicates a mechanism by which HSV-1 viral genes in a quiescent or latent state may become induced by cellular DNA damage in neuronal cells to facilitate productive infection. PMID:23354549

  10. DNA damage tolerance: a double-edged sword guarding the genome

    PubMed Central

    Ghosal, Gargi; Chen, Junjie

    2013-01-01

    Preservation of genome integrity is an essential process for cell homeostasis. During the course of life of a single cell, the genome is constantly damaged by endogenous and exogenous agents. To ensure genome stability, cells use a global signaling network, namely the DNA damage response (DDR) to sense and repair DNA damage. DDR senses different types of DNA damage and coordinates a response that includes activation of transcription, cell cycle control, DNA repair pathways, apoptosis, senescence, and cell death. Despite several repair mechanisms that repair different types of DNA lesions, it is likely that the replication machinery would still encounter lesions that are mis-repaired or not repaired. Replication of damaged genome would result in high frequency of fork collapse and genome instability. In this scenario, the cells employ the DNA damage tolerance (DDT) pathway that recruits a specialized low fidelity translesion synthesis (TLS) polymerase to bypass the lesions for repair at a later time point. Thus, DDT is not a repair pathway per se, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and preventing genome instability. Paradoxically, DDT process is also associated with increased mutagenesis, which can in turn drive the cell to cancer development. Thus, DDT process functions as a double-edged sword guarding the genome. In this review, we will discuss the replication stress induced DNA damage-signaling cascade, the stabilization and rescue of stalled replication forks by the DDT pathway and the effect of the DDT pathway on cancer. PMID:24058901

  11. Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study

    SciTech Connect

    Muniz, Juan F. McCauley, Linda; Scherer, J.; Lasarev, M.; Koshy, M.; Kow, Y.W.; Nazar-Stewart, Valle; Kisby, G.E.

    2008-02-15

    Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers and applicators (p < 0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects.

  12. Olaquindox induces DNA damage via the lysosomal and mitochondrial pathway involving ROS production and p53 activation in HEK293 cells.

    PubMed

    Yang, Yang; Jiang, Liping; She, Yan; Chen, Min; Li, Qiujuan; Yang, Guang; Geng, Chengyan; Tang, Liyun; Zhong, Laifu; Jiang, Lijie; Liu, Xiaofang

    2015-11-01

    Olaquindox (OLA) is a potent antibacterial agent used as a feed additive and growth promoter. In this study, the genotoxic potential of OLA was investigated in the human embryonic kidney cell line 293 (HEK293). Results showed that OLA caused significant increases of DNA migration. Lysosomal membrane permeability and mitochondrial membrane potential were reduced after treatment with OLA. OLA was shown to induce ROS production and GSH depletion. The expression of p53 protein is increased in cells incubated with OLA. The activation of p53 and ATM gene was assessed by exposure to OLA. Furthermore, NAC reduced DNA migration, ROS formation, GSH depletion and the expression of the p53 protein and gene. And desipramine significantly decreased AO fluorescence intensity and the expression of the p53 protein and gene. These results support the assumption that OLA exerted genotoxic effects and induced DNA strand breaks in HEK293 cells, possibly through lysosomal-mitochondrial pathway involving ROS production and p53 activation.

  13. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest

    PubMed Central

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P.; Chow, Vincent T.K.

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  14. 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

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

    PubMed

    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

  16. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest.

    PubMed

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P; Chow, Vincent T K

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  17. Negative control of CSL gene transcription by stress/DNA damage response and p53.

    PubMed

    Menietti, Elena; Xu, Xiaoying; Ostano, Paola; Joseph, Jean-Marc; Lefort, Karine; Dotto, G Paolo

    2016-07-01

    CSL is a key transcriptional repressor and mediator of Notch signaling. Despite wide interest in CSL, mechanisms responsible for its own regulation are little studied. CSL down-modulation in human dermal fibroblasts (HDFs) leads to conversion into cancer associated fibroblasts (CAF), promoting keratinocyte tumors. We show here that CSL transcript levels differ among HDF strains from different individuals, with negative correlation with genes involved in DNA damage/repair. CSL expression is negatively regulated by stress/DNA damage caused by UVA, Reactive Oxygen Species (ROS), smoke extract, and doxorubicin treatment. P53, a key effector of the DNA damage response, negatively controls CSL gene transcription, through suppression of CSL promoter activity and, indirectly, by increased p21 expression. CSL was previously shown to bind p53 suppressing its activity. The present findings indicate that p53, in turn, decreases CSL expression, which can serve to enhance p53 activity in acute DNA damage response of cells.

  18. Link between DNA damage and centriole disengagement/reduplication in untransformed human cells †

    PubMed Central

    Douthwright, Stephen; Sluder, Greenfield

    2014-01-01

    The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 hours, though 14% of the cells eventually go through mitosis in that time. By 72 hours after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53 dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. PMID:24532022

  19. Fine-mapping of DNA damage and repair in specific genomic segments.

    PubMed Central

    Govan, H L; Valles-Ayoub, Y; Braun, J

    1990-01-01

    The susceptibility of various genomic regions to DNA damage and repair is heterogeneous. While this can be related to factors such as primary sequence, physical conformation, and functional status, the exact mechanisms involved remain unclear. To more precisely define the key features of a genomic region targeted for these processes, a useful tool would be a method for fine-mapping gene-specific DNA damage and repair in vivo. Here, a polymerase chain reaction-based assay is described for measuring DNA damage and repair in small (less than 500 bp) genomic segments of three transcriptionally active but functionally distinct loci (rearranged immunoglobulin heavy chain variable region [Ig VDJ], low-density lipoprotein receptor gene, and N-ras proto-oncogene) in human tonsillar B lymphocytes. Analysis of ultraviolet (254 nm)-induced DNA damage revealed single-hit kinetics and a similar level of sensitivity (D50% approximately 6000 joule/m2) in all three regions, indicating that a single photoproduct was sufficient to fully block PCR amplification. A similar time period per unit length was required for repair of this DNA damage (average t1/2 per fragment length = 23.5 seconds per bp). DNA damage and repair was also detectable with the base adducting agent, 4-nitroquinoline-1-oxide. However, in this case IgVDJ differed from segments within the other two loci by its relative inaccessibility to alkylation. This assay thus permits high-resolution mapping of DNA damage and repair activity. Images PMID:2115669

  20. Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

    PubMed

    Douthwright, Stephen; Sluder, Greenfield

    2014-10-01

    The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations.

  1. 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.

  2. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  3. DNA damage is a late event in resveratrol-mediated inhibition of Escherichia coli.

    PubMed

    Subramanian, Mahesh; Soundar, Swetha; Mangoli, Suhas

    2016-07-01

    Resveratrol is an important phytoalexin notable for a wide variety of beneficial activities. Resveratrol has been reported to be active against various pathogenic bacteria. However, it is not clear at the molecular level how this important activity is manifested. Resveratrol has been reported to bind to cupric ions and reduce it. In the process, it generates copper-peroxide complex and reactive oxygen species (ROS). Due to this ability, resveratrol has been shown to cleave plasmid DNA in several studies. To this end, we envisaged DNA damage to play a role in resveratrol mediated inhibition in Escherichia coli. We employed DNA damage repair deficient mutants from keio collection to demonstrate the hypersensitive phenotype upon resveratrol treatment. Analysis of integrity and PCR efficiency of plasmid DNA from resveratrol-treated cells revealed significant DNA damage after 6 h or more compared to DNA from vehicle-treated cells. RAPD-PCR was performed to demonstrate the damage in genomic DNA from resveratrol-treated cells. In addition, in situ DNA damage was observed under fluorescence microscopy after resveratrol treatment. Further resveratrol treatment resulted in cell cycle arrest of significant fraction of population revealed by flow cytometry. However, a robust induction was not observed in phage induction assay and induction of DNA damage response genes quantified by promoter fused fluorescent tracker protein. These observations along with our previous observation that resveratrol induces membrane damage in E. coli at early time point reveal, DNA damage is a late event, occurring after a few hours of treatment. PMID:27021971

  4. HTLV-1 Tax protein sensitizes cells to apoptotic cell death induced by DNA damaging agents.

    PubMed

    Kao, S Y; Lemoine, F J; Mariott, S J

    2000-04-27

    Transient HTLV-1 Tax expression suppresses cellular nucleotide excision repair, and this effect correlates with Tax transactivation of the proliferating cell nuclear antigen promoter. The inability to repair DNA damage typically induces apoptotic cell death. Therefore, we investigated the effect of Tax-mediated suppression of DNA repair on apoptosis in stable Tax-expressing cells. Constitutive Tax expression reduced cellular nucleotide excision repair activity compared with parental and control cells. Tax-expressing cells were also more sensitive to apoptosis induced by DNA damaging agents than control cells. Even though Tax-expressing cells displayed reduced DNA repair, they showed increased DNA replication following UV damage. These results suggest that Tax suppresses the cell's ability to repair DNA damage and stimulates DNA replication even in the presence of damage. The inability to repair DNA damage is likely to stimulate apoptotic cell death in the majority of Tax-expressing cells while the ability to promote DNA replication may also allow the survival of a small population of cells. We propose that together these effects contribute to the monoclonal nature and low efficiency of HTLV-1 transformation.

  5. The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

    PubMed Central

    De Zio, D; Bordi, M; Tino, E; Lanzuolo, C; Ferraro, E; Mora, E; Ciccosanti, F; Fimia, G M; Orlando, V; Cecconi, F

    2011-01-01

    Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival. PMID:20966962

  6. p53 and Cell Cycle Effects After DNA Damage

    PubMed Central

    Senturk, Emir; Manfredi, James J.

    2016-01-01

    Flow cytometry, a valuable technique that employs the principles of light scattering, light excitation, and emission of fluorochrome molecules, can be used to assess the cell cycle position of individual cells based on DNA content. After the permeabilization of cells, the DNA can be stained with a fluorescent dye. Cells which have a 2N amount of DNA can be distinguished from cells with a 4N amount of DNA, making flow cytometry a very useful tool for the analysis of cell cycle checkpoints following DNA damage. A critical feature of the cellular response to DNA damage is the ability to pause and repair the damage so that consequential mutations are not passed along to daughter generations of cells. If cells arrest prior to DNA replication, they will contain a 2N amount of DNA, whereas arrest after replication but before mitosis will result in a 4N amount of DNA. Using this technique, the role that p53 plays in cell cycle checkpoints following DNA damage can be evaluated based on changes in the profile of the G1, S, and G2/M phases of the cell cycle. PMID:23150436

  7. Senescence of Primary Amniotic Cells via Oxidative DNA Damage

    PubMed Central

    Menon, Ramkumar; Boldogh, Istvan; Urrabaz-Garza, Rheanna; Polettini, Jossimara; Syed, Tariq Ali; Saade, George R.; Papaconstantinou, John; Taylor, Robert N.

    2013-01-01

    Objective Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells. Methods Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2′7′-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19arf. Expression of senescence-associated β-galectosidase (SAβ-gal) was used to confirm cell ageing in situ. Results ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19Arf were not significantly affected by NAC. Conclusions The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB. PMID:24386195

  8. Hypersensitivity to DNA damage in antephase as a safeguard for genome stability.

    PubMed

    Feringa, Femke M; Krenning, Lenno; Koch, André; van den Berg, Jeroen; van den Broek, Bram; Jalink, Kees; Medema, René H

    2016-01-01

    Activation of the DNA-damage response can lead to the induction of an arrest at various stages in the cell cycle. These arrests are reversible in nature, unless the damage is too excessive. Here we find that checkpoint reversibility is lost in cells that are in very late G2, but not yet fully committed to enter mitosis (antephase). We show that antephase cells exit the cell cycle and enter senescence at levels of DNA damage that induce a reversible arrest in early G2. We show that checkpoint reversibility critically depends on the presence of the APC/C inhibitor Emi1, which is degraded just before mitosis. Importantly, ablation of the cell cycle withdrawal mechanism in antephase promotes cell division in the presence of broken chromosomes. Thus, our data uncover a novel, but irreversible, DNA-damage response in antephase that is required to prevent the propagation of DNA damage during cell division. PMID:27561326

  9. Hypersensitivity to DNA damage in antephase as a safeguard for genome stability.

    PubMed

    Feringa, Femke M; Krenning, Lenno; Koch, André; van den Berg, Jeroen; van den Broek, Bram; Jalink, Kees; Medema, René H

    2016-01-01

    Activation of the DNA-damage response can lead to the induction of an arrest at various stages in the cell cycle. These arrests are reversible in nature, unless the damage is too excessive. Here we find that checkpoint reversibility is lost in cells that are in very late G2, but not yet fully committed to enter mitosis (antephase). We show that antephase cells exit the cell cycle and enter senescence at levels of DNA damage that induce a reversible arrest in early G2. We show that checkpoint reversibility critically depends on the presence of the APC/C inhibitor Emi1, which is degraded just before mitosis. Importantly, ablation of the cell cycle withdrawal mechanism in antephase promotes cell division in the presence of broken chromosomes. Thus, our data uncover a novel, but irreversible, DNA-damage response in antephase that is required to prevent the propagation of DNA damage during cell division.

  10. Hypersensitivity to DNA damage in antephase as a safeguard for genome stability

    PubMed Central

    Feringa, Femke M.; Krenning, Lenno; Koch, André; van den Berg, Jeroen; van den Broek, Bram; Jalink, Kees; Medema, René H.

    2016-01-01

    Activation of the DNA-damage response can lead to the induction of an arrest at various stages in the cell cycle. These arrests are reversible in nature, unless the damage is too excessive. Here we find that checkpoint reversibility is lost in cells that are in very late G2, but not yet fully committed to enter mitosis (antephase). We show that antephase cells exit the cell cycle and enter senescence at levels of DNA damage that induce a reversible arrest in early G2. We show that checkpoint reversibility critically depends on the presence of the APC/C inhibitor Emi1, which is degraded just before mitosis. Importantly, ablation of the cell cycle withdrawal mechanism in antephase promotes cell division in the presence of broken chromosomes. Thus, our data uncover a novel, but irreversible, DNA-damage response in antephase that is required to prevent the propagation of DNA damage during cell division. PMID:27561326

  11. Clusters of DNA damage induced by ionizing radiation: formation of short DNA fragments. II. Experimental detection

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

    The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.

  12. 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

  13. The DNA damage response: the omics era and its impact

    PubMed Central

    Derks, Kasper W.J.; Hoeijmakers, Jan H.J.; Pothof, Joris

    2014-01-01

    The emergence of high density technologies monitoring the genome, transcriptome and proteome in relation to genotoxic stress have tremendously enhanced our knowledge on global responses and dynamics in the DNA damage response, including its relation with cancer and aging. Moreover, ‘-omics’ technologies identified many novel factors, their post-translational modifications, pathways and global responses in the cellular response to DNA damage. Based on omics, it is currently estimated that thousands of gene(product)s participate in the DNA damage response, recognizing complex networks that determine cell fate after damage to the most precious cellular molecule, DNA. The development of next generation sequencing technology and associated specialized protocols can quantitatively monitor RNA and DNA at unprecedented single nucleotide resolution. In this review we will discuss the contribution of omics technologies and in particular next generation sequencing to our understanding of the DNA damage response and the future prospective of next generation sequencing, its single cell application and omics dataset integration in unraveling intricate DNA damage signaling networks. PMID:24794401

  14. Antioxidant and DNA damage protection potentials of selected phenolic acids.

    PubMed

    Sevgi, Kemal; Tepe, Bektas; Sarikurkcu, Cengiz

    2015-03-01

    In this study, ten different phenolic acids (caffeic, chlorogenic, cinnamic, ferulic, gallic, p-hydroxybenzoic, protocatechuic, rosmarinic, syringic, and vanillic acids) were evaluated for their antioxidant and DNA damage protection potentials. Antioxidant activity was evaluated by using four different test systems named as β-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. In all test systems, rosmarinic acid showed the maximum activity potential, while protocatechuic acid was determined as the weakest antioxidant in β-carotene bleaching, DPPH free radical scavenging, and chelating effect assays. Phenolic acids were also screened for their protective effects on pBR322 plasmid DNA against the mutagenic and toxic effects of UV and H2O2. Ferulic acid was found as the most active phytochemical among the others. Even at the lowest concentration value (0.002 mg/ml), ferulic acid protected all of the bands in the presence of H2O2 and UV. It is followed by caffeic, rosmarinic, and vanillic acids. On the other hand, cinnamic acid (at 0.002 mg/ml), gallic acid (at 0.002 mg/ml), p-hydroxybenzoic acid (at 0.002 and 0.004 mg/ml), and protocatechuic acid (at 0.002 and 0.004 mg/ml) could not protect plasmid DNA. PMID:25542528

  15. Chemopreventive activity of compounds extracted from Casearia sylvestris (Salicaceae) Sw against DNA damage induced by particulate matter emitted by sugarcane burning near Araraquara, Brazil

    SciTech Connect

    Prieto, A.M.; Santos, A.G.; Csipak, A.R.; Caliri, C.M.; Silva, I.C.; Arbex, M.A.; Silva, F.S.; Marchi, M.R.R.

    2012-12-15

    Ethanolic extract of Casearia sylvestris is thought to be antimutagenic. In this study, we attempted to determine whether this extract and casearin X (a clerodane diterpene from C. sylvestris) are protective against the harmful effects of airborne pollutants from sugarcane burning. To that end, we used the Tradescantia micronucleus test in meiotic pollen cells of Tradescantia pallida, the micronucleus test in mouse bone marrow cells, and the comet assay in mouse blood cells. The mutagenic compound was total suspended particulate (TSP) from air. For the Tradescantia micronucleus test, T. pallida cuttings were treated with the extract at 0.13, 0.25, or 0.50 mg/ml. Subsequently, TSP was added at 0.3 mg/ml, and tetrads from the inflorescences were examined for micronuclei. For the micronucleus test in mouse bone marrow cells and the comet assay in mouse blood cells, Balb/c mice were treated for 15 days with the extract—3.9, 7.5, or 15.0 mg/kg body weight (BW)—or with casearin X—0.3, 0.25, or 1.2 mg/kg BW—after which they received TSP (3.75 mg/kg BW). In T. pallida and mouse bone marrow cells, the extract was antimutagenic at all concentrations tested. In mouse blood cells, the extract was antigenotoxic at all concentrations, whereas casearin X was not antimutagenic but was antigenotoxic at all concentrations. We conclude that C. sylvestris ethanolic extract and casearin X protect DNA from damage induced by airborne pollutants from sugarcane burning. -- Highlights: ► We assessed DNA protection of C. sylvestris ethanolic extract. ► We assessed DNA protection of casearin X. ► We used Tradescantia pallida micronucleus test as screening. ► We used comet assay and micronucleus test in mice. ► The compounds protected DNA against sugar cane burning pollutants.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Kai, Mihoko

    2016-01-01

    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. Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response

    PubMed Central

    Moreli, Jusciele B.; Santos, Janine H.; Lorenzon-Ojea, Aline Rodrigues; Corrêa-Silva, Simone; Fortunato, Rodrigo S.; Rocha, Clarissa Ribeiro; Rudge, Marilza V.; Damasceno, Débora C.; Bevilacqua, Estela; Calderon, Iracema M.

    2016-01-01

    Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ and FEN1). A diabetes-like environment was unable to induce apoptosis in the umbilical cord blood cells. Conclusions: Our data show relevant asymmetry between maternal and fetal blood cell susceptibility to DNA damage and apoptosis induction. Maternal cells seem to be more predisposed to changes in an adverse glucose environment. This may be due to differential ability in upregulating multiple genes involved in the activation of DNA repair response, especially the BER mechanism. However if this study shows a more effective adaptive response by the fetal organism, it also calls for

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Sun, Xin; Fu, Kai; Hodgson, Andrea; Wier, Eric M; Wen, Matthew G; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y; Wan, Fengyi

    2016-09-01

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

  1. DNA Damage-induced Reactive Oxygen Species (ROS) Stress Response in Saccharomyces cerevisiae

    PubMed Central

    Rowe, Lori A.; Degtyareva, Natalya; Doetsch, Paul W.

    2008-01-01

    Cells are exposed to both endogenous and exogenous sources of reactive oxygen species (ROS). At high levels, ROS can lead to impaired physiological function through cellular damage of DNA, proteins, lipids, and other macromolecules, which can lead to certain human pathologies including cancers, neurodegenerative disorders, and cardiovascular disease, as well as aging. We have employed Saccharomyces cerevisiae as a model system to examine the levels and types of ROS that are produced in response to DNA damage in isogenic strains with different DNA repair capacities. We find that when DNA damage is introduced into cells from exogenous or endogenous sources there is an increase in the amount of intracellular ROS which is not directly related to cell death. We have examined the spectrum of ROS in order to elucidate its role in the cellular response to DNA damage. As an independent verification of the DNA damage-induced ROS response, we show that a major activator of the oxidative stress response, Yap1, relocalizes to the nucleus following exposure to the DNA alkylating agent methyl methanesulfonate. Our results indicate that the DNA damage-induced increase in intracellular ROS levels is a generalized stress response that is likely to function in various signaling pathways. PMID:18708137

  2. 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.

  3. Oxidative status and DNA damage in chidren with marasmic malnutrition.

    PubMed

    Celik, Maruf; Sermatov, Kabil; Abuhandan, Mahmut; Zeyrek, Dost; Kocyigit, Abdurrahim; Iscan, Akin

    2012-05-01

    Malnutrition as a lack of several substances containing antioxidants such as vitamins and micronutrients, while showing a predisposition for lipid peroxidation and DNA damage, is also characterized by a slowing down of the metabolic processes, which may then have protective properties against DNA damage due to a reduction in endogenous free radical production. This study aimed to examine the oxidative status and DNA damage in cases of marasmus. The study comprised 28 infants aged 6-24 months with marasmus only and 28 age-matched healthy infants. DNA damage was examined by the alkali single cell electrophoresis method (Comet assay) on mononuclear leukocytes. The total oxidant status (TOS) and total antioxidant status (TAS) were measured by colormetric auto-analyzer and the oxidative stress index (OSI) was calculated. The TOS, TAS, and OSI levels of the patient group were found to be significantly lower compared to the control group (P < 0.01, P < 0.01, P < 0.01, respectively). No statistically significant difference was found between the two groups in terms of mononuclear leukocyte DNA damage (P > 0.05). The findings of this study showed that in marasmus cases, the oxidative and antioxidative processes, which have a counteractive effect, decreased together. The other results of the study indicate that there is no increase in DNA damage in marasmus cases.

  4. DNA damage recognition in the rat zygote following chronic paternal cyclophosphamide exposure.

    PubMed

    Barton, Tara S; Robaire, Bernard; Hales, Barbara F

    2007-12-01

    The detrimental effects of preconceptional paternal exposure to the alkylating anticancer agent, cyclophosphamide, include aberrant epigenetic programming, dysregulated zygotic gene activation, and abnormalities in the offspring that are transmitted to the next generation. The adverse developmental consequences of genomic instabilities transmitted via the spermatozoon emphasize the need to elucidate the mechanisms by which the early embryo recognizes DNA damage in the paternal genome. Little information exists on DNA damage detection in the zygote. We assessed the impact of paternal cyclophosphamide exposure on phosphorylated H2AX (gammaH2AX) and poly(ADP-ribose) polymerase-1(PARP-1), biomarkers of DNA damage, to determine the capacity in the rat zygote to recognize genomic damage and initiate a response to DNA lesions. An amplified biphasic gammaH2AX response was triggered in the paternal pronucleus in zygotes sired by drug-treated males; the maternal genome was not affected. PARP-1 immunoreactivity was substantially elevated in both parental genomes, coincident with the second phase of gammaH2AX induction in embryos sired by cyclophosphamide-exposed spermatozoa. Thus, paternal exposure to a DNA damaging agent rapidly activates signals implemental for DNA damage recognition in the zygote. Inefficient repair of DNA lesions may lead to persistent alterations of the histone code and chromatin integrity, resulting in aberrant embryogenesis. We propose that the response of the early embryo to disturbances in spermatozoal genomic integrity plays a vital role in determining its outcome.

  5. Conformation and dynamics of normal and damaged DNA.

    PubMed

    Rachofsky, E L; Ross, J B; Osman, R

    2001-12-01

    The genetic information that determines the structure and function of living organisms is encoded in the nucleotide sequence of double-stranded DNA molecules. Despite an apparent structural homogeneity displayed by DNA, subtle local variations in structure and dynamics are functionally significant. Short sequences exhibit specificity for regulatory and catalytic proteins, which mediate fundamental processes necessary to the survival of the cell. However, the molecular basis for specific recognition is still incompletely understood. The "indirect readout" mechanism suggests that the relative propensity of DNA to undergo structural deformations induced by the protein contributes to specific protein-DNA recognition. Although the hypothesis was originally formulated to explain recognition of specific nucleic acid sequences by DNA-binding proteins, it may have particular application to the recognition of DNA damage, because damaged sites in DNA have different equilibrium structure and dynamics from undamaged DNA. In this work, we review the approaches that we took to investigate the questions of sequence- and damage-dependent structure and dynamics of DNA. We describe a statistical thermodynamic model that relates DNA configurational flexibility to sequence-specific protein-DNA binding. The model provides a theoretical basis for interpreting experimental measurements of DNA dynamics. We describe results from MCSCF calculations of the excited states of 2-aminopurine (2AP), which provide the theoretical basis for the intramolecular mechanism of quenching as well as the effect of environment on this process. We then describe our investigations of the effect of stacking, base pairing, and base dynamics on the fluorescence of 2-AP in model systems, which allow us to develop the relationships between steady-state and time-resolved fluorescence parameters on the one hand and local structural and dynamic properties of DNA on the other hand. Finally, we describe the application

  6. 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. PMID:17132903

  7. Utilization of an automated pipetting system in the cell line-based screening of the activity of a DNA-damaging anti-tumour drug.

    PubMed

    Suchánková, T; Ovesná, P; Samadder, P; Souček, K

    2014-01-01

    The principles of large screening strategies, which are developed by industrial companies, have been recently adopted by researchers in the fields of molecular biology and oncology as invaluable tools for translational medicine. The declining costs of laboratory robotic machines have allowed high-throughput screening to become more available to academic centres with limited resources. Here, we describe how a robotic conventional liquid handling system could be used on a daily basis in laboratories to obtain consistent and reproducible results. Our approach allowed us to quickly screen a panel of more than 20 tumorigenic and non-tumorigenic cell lines for their responses to hydroxyurea, which is a DNA-damaging anticancer therapeutic drug. The format of 384-well microplates was used for manual cell seeding, and the effect of hydroxyurea was screened at multiple concentrations. The fluorescence-based CyQuant assay was employed as the readout method to analyse the cellular DNA content. The effectiveness of our approach was demonstrated in the experimental results.

  8. Oxidative DNA damage induced by aminoacetone, an amino acid metabolite.

    PubMed

    Hiraku, Y; Sugimoto, J; Yamaguchi, T; Kawanishi, S

    1999-05-01

    We investigated DNA damage induced by aminoacetone, a metabolite of threonine and glycine. Pulsed-field gel electrophoresis revealed that aminoacetone caused cellular DNA cleavage. Aminoacetone increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human cultured cells in a dose-dependent manner. The formation of 8-oxodG in calf thymus DNA increased due to aminoacetone only in the presence of Cu(II). DNA ladder formation was observed at higher concentrations of aminoacetone than those causing DNA cleavage. Flow cytometry showed that aminoacetone enhanced the generation of hydrogen peroxide (H2O2) in cultured cells. Aminoacetone caused damage to 32P-5'-end-labeled DNA fragments, obtained from the human c-Ha-ras-1 and p53 genes, at cytosine and thymine residues in the presence of Cu(II). Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. Analysis of the products generated from aminoacetone revealed that aminoacetone underwent Cu(II)-mediated autoxidation in two different pathways: the major pathway in which methylglyoxal and NH+4 are generated and the minor pathway in which 2,5-dimethylpyrazine is formed through condensation of two molecules of aminoacetone. These findings suggest that H2O2 generated by the autoxidation of aminoacetone reacts with Cu(I) to form reactive species capable of causing oxidative DNA damage.

  9. Nutritional and lifestyle determinants of DNA oxidative damage: a study in a Mediterranean population.

    PubMed

    Giovannelli, Lisa; Saieva, Calogero; Masala, Giovanna; Testa, Giovanna; Salvini, Simonetta; Pitozzi, Vanessa; Riboli, Elio; Dolara, Piero; Palli, Domenico

    2002-09-01

    In order to evaluate dietary and lifestyle determinants of oxidative DNA damage we used a modification of the 'comet assay' (single cell alkaline gel electrophoresis), with the fpg enzyme (formamidopyrimidine DNA glycosilase), to measure the basal level of DNA oxidation in peripheral lymphocytes donated by 71 healthy adults living in Florence, Italy. Detailed information about dietary and lifestyle habits was collected by two validated and standardized questionnaires; we also measured plasma concentrations of selected micro-nutrients (six carotenoids, retinol, alpha- and gamma-tocopherol). DNA damage, measured as percent DNA migrated in the comet tail (mean 4.67%, interquartile range 2.36-6.62%), was not associated with gender, age, weight, body mass index, physical activity or smoking history. A positive correlation with height and period of blood sampling emerged: DNA damage tended to be higher among taller subjects (P = 0.02) and in samples obtained in summer months (P = 0.02). Multivariate analyses showed a positive association with coffee (P = 0.01) and tomato consumption (P = 0.05). Instead, the consumption of cruciferous vegetables tended to be negatively associated with oxidative damage (P = 0.09). Furthermore, a positive non-significant association between the consumption of total vegetables and fresh fruit and DNA damage emerged (P = 0.08 and P = 0.10, respectively). The estimated intake of simple sugars showed a strong positive association with oxidative DNA damage (P = 0.01), while vitamin E showed a borderline positive association (P = 0.06). The plasma levels of several micro-nutrients did not appear to influence DNA damage. Our results, although based on a relatively small group of subjects, indicate that individual dietary and lifestyle habits only modestly affect the levels of lymphocyte DNA oxidation and suggest that specific dietary patterns, rich in fresh fruit and vegetables, are not clearly related to decreased oxidative damage in peripheral

  10. 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.

  11. T7 replisome directly overcomes DNA damage

    PubMed Central

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

    2015-01-01

    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. PMID:26675048

  12. Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

    PubMed

    Biswas, Jaydip; Sinha, Dona; Mukherjee, Sutapa; Roy, Soumi; Siddiqi, Maqsood; Roy, Madhumita

    2010-06-01

    Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic.

  13. CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development.

    PubMed

    Wang, HaiYang; Kim, Nam-Hyung

    2016-08-01

    Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression. PMID:27307074

  14. Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

    PubMed

    Biswas, Jaydip; Sinha, Dona; Mukherjee, Sutapa; Roy, Soumi; Siddiqi, Maqsood; Roy, Madhumita

    2010-06-01

    Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic. PMID:20056736

  15. The DNA-damage response in human biology and disease

    PubMed Central

    Jackson, Stephen P.; Bartek, Jiri

    2010-01-01

    The prime objective for every life-form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which impact a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management. PMID:19847258

  16. UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation

    PubMed Central

    Brand, Marjorie; Moggs, Jonathan G.; Oulad-Abdelghani, Mustapha; Lejeune, Fabrice; Dilworth, F.Jeffrey; Stevenin, James; Almouzni, Geneviève; Tora, Làszlò

    2001-01-01

    Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAFIIs). In the presence of TBP-free TAFII complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification. PMID:11406595

  17. Real-time damage monitoring of irradiated DNA.

    PubMed

    Pješčić, Ilija; Tranter, Collin A; Haywood, James C; Paidipalli, Manasa; Ganveer, Ankur; Haywood, Stratton E; Tham, Jessica; Crews, Niel D

    2011-09-01

    This article presents a microfluidic technique for the real-time analysis of DNA damage due to radiation exposure. A continuous-flow spatial melting analysis was performed every three seconds on a sample of isolated DNA while it was being irradiated. The formation of photoproducts being caused by the UV-C radiation was monitored during the process. Cumulative damage produced distinct changes in the DNA melting curves, characterized by a shifting and broadening of the melting peaks. The design of the microfluidic device, the experimental procedure, and the analysis algorithm and interactive GUI are discussed herein. In addition, the advantages of this system are correlated to specific needs of related scientific studies, such as the investigation of sequence-specific damage susceptibility and the characterization of exposure-damage nonlinearities.

  18. LEM-3 - A LEM domain containing nuclease involved in the DNA damage response in C. elegans.

    PubMed

    Dittrich, Christina M; Kratz, Katja; Sendoel, Ataman; Gruenbaum, Yosef; Jiricny, Josef; Hengartner, Michael O

    2012-01-01

    The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage - possibly by promoting the reorganization of damaged chromatin. PMID:22383942

  19. 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

    2013-12-01

    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.

  20. 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

    2013-12-01

    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. PMID:24248361

  1. Bacterial natural transformation by highly fragmented and damaged DNA

    PubMed Central

    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

    2013-01-01

    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. PMID:24248361

  2. [Photoreactivating Activity of Bioluminescence: Repair of UV-damaged DNA of Escherichia coli Occurs with Assistance of lux-Genes of Marine Bacteria].

    PubMed

    Zavilgelsky, G B; Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K Ss

    2015-01-01

    The UV resistance of luminescent bacteria Escherichia coli AB1886 uvrA6 (pLeo1) containing the plasmid with luxCDABE genes of marine bacteria Photobacterium leiognathi is approximately two times higher than the UV resistance of non-luminous bacteria E. coli AB1886 uvrA6. Introduction of phr::kan(r) mutations (a defect in the functional activity of photolyase) into the genome of E. coli AB1886 uvrA6 (pLeo1) completely removes the high UV resistance of the cells. Therefore, photoreactivation that involves bacterial photolyase contributes mainly to the bioluminescence-induced DNA repair. It is shown that photoreactivating activity of bioluminescence of P. leiognathi is about 2.5 times lower compared with that one induced by a light source with λ > 385 nm. It is also shown that an increase in the bioluminescence intensity, induced by UV radiation in E. coli bacterial cells with a plasmid containing the luxCD ABE genes under RecA-LexA-regulated promoters, occurs only 25-30 min later after UV irradiation of cells and does not contribute to DNA repair. A quorum sensing regulatory system is not involved in the DNA repair by photolyase.

  3. Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Senoo, Takanori; Yamanaka, Mayumi; Nakamura, Atori; Terashita, Tomoki; Kawano, Shinji; Ikeda, Shogo

    2016-08-01

    Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging.

  4. Non-coding RNAs: an emerging player in DNA damage response.

    PubMed

    Zhang, Chunzhi; Peng, Guang

    2015-01-01

    Non-coding RNAs play a crucial role in maintaining genomic stability which is essential for cell survival and preventing tumorigenesis. Through an extensive crosstalk between non-coding RNAs and the canonical DNA damage response (DDR) signaling pathway, DDR-induced expression of non-coding RNAs can provide a regulatory mechanism to accurately control the expression of DNA damage responsive genes in a spatio-temporal manner. Mechanistically, DNA damage alters expression of a variety of non-coding RNAs at multiple levels including transcriptional regulation, post-transcriptional regulation, and RNA degradation. In parallel, non-coding RNAs can directly regulate cellular processes involved in DDR by altering expression of their targeting genes, with a particular emphasis on miRNAs and lncRNAs. MiRNAs are required for almost every aspect of cellular responses to DNA damage, including sensing DNA damage, transducing damage signals, repairing damaged DNA, activating cell cycle checkpoints, and inducing apoptosis. As for lncRNAs, they control transcription of DDR relevant gene by four different regulatory models, including signal, decoy, guide, and scaffold. In addition, we also highlight potential clinical applications of non-coding RNAs as biomarkers and therapeutic targets for anti-cancer treatments using DNA-damaging agents including radiation and chemotherapy. Although tremendous advances have been made to elucidate the role of non-coding RANs in genome maintenance, many key questions remain to be answered including mechanistically how non-coding RNA pathway and DNA damage response pathway is coordinated in response to genotoxic stress.

  5. Higher order DNA structure and radiation damage

    SciTech Connect

    Oleinick, N.L.; Chiu, S.M.; Xue, L.Y.; Friedman, L.R.; Balasubramaniam, U.

    1995-12-31

    Work until now has implicated chromatin structure and/or chromatin proteins as both radioprotectors of cellular DNA for double-strand breaks (DSB) induction and substrates for DNA-protein crosslinks (DPC) production. In the present study, the authors have attempted further to differentiate between the possible roles of chromatin proteins by reconstitution of chromatin. One or more hypertonic salt extracts or commercial histones were added back to dehistonized nuclei, following which the preparations were irradiated and the yields of DPC and DSB were measured.

  6. The comet assay for DNA damage and repair: principles, applications, and limitations.

    PubMed

    Collins, Andrew R

    2004-03-01

    The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage. PMID:15004294

  7. The human DEK oncogene regulates DNA damage response signaling and repair

    PubMed Central

    Kavanaugh, Gina M.; Wise-Draper, Trisha M.; Morreale, Richard J.; Morrison, Monique A.; Gole, Boris; Schwemberger, Sandy; Tichy, Elisia D.; Lu, Lu; Babcock, George F.; Wells, James M.; Drissi, Rachid; Bissler, John J.; Stambrook, Peter J.; Andreassen, Paul R.; Wiesmüller, Lisa; Wells, Susanne I.

    2011-01-01

    The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair. PMID:21653549

  8. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    SciTech Connect

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-12-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA{sup III}); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA{sup III} [URO-MSC(+)] and after subsequent removal of MMA{sup III} [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA{sup III}, URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA{sup III}. URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA{sup III} exposure, suggesting the presence of MMA{sup III} is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA{sup III} results in: the induction of DNA damage that remains elevated upon removal of MMA{sup III}; increased levels of ROS that play a role in MMA{sup III} induced-DNA damage; and decreased PARP activity in the presence of MMA{sup III}.

  9. 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

  10. 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

  11. Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker.

    PubMed

    Havran, Ludek; Vacek, Jan; Cahová, Katerina; Fojta, Miroslav

    2008-07-01

    This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.

  12. Common Fragile Sites: Genomic Hotspots of DNA Damage and Carcinogenesis

    PubMed Central

    Ma, Ke; Qiu, Li; Mrasek, Kristin; Zhang, Jun; Liehr, Thomas; Quintana, Luciana Gonçalves; Li, Zheng

    2012-01-01

    Genomic instability, a hallmark of cancer, occurs preferentially at specific genomic regions known as common fragile sites (CFSs). CFSs are evolutionarily conserved and late replicating regions with AT-rich sequences, and CFS instability is correlated with cancer. In the last decade, much progress has been made toward understanding the mechanisms of chromosomal instability at CFSs. However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci. Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis. The identification of FATS may inspire more discoveries of other uncharacterized CACGs. Further elucidation of the biological functions and clinical significance of CACGs may be exploited for cancer biomarkers and therapeutic benefits. PMID:23109895

  13. Antimalarial action of artesunate involves DNA damage mediated by reactive oxygen species.

    PubMed

    Gopalakrishnan, Anusha M; Kumar, Nirbhay

    2015-01-01

    Artemisinin-based combination therapy (ACT) is the recommended first-line treatment for Plasmodium falciparum malaria. It has been suggested that the cytotoxic effect of artemisinin is mediated by free radicals followed by the alkylation of P. falciparum proteins. The endoperoxide bridge, the active moiety of artemisinin derivatives, is cleaved in the presence of ferrous iron, generating reactive oxygen species (ROS) and other free radicals. However, the emergence of resistance to artemisinin in P. falciparum underscores the need for new insights into the molecular mechanisms of antimalarial activity of artemisinin. Here we show that artesunate (ART) induces DNA double-strand breaks in P. falciparum in a physiologically relevant dose- and time-dependent manner. DNA damage induced by ART was accompanied by an increase in the intracellular ROS level in the parasites. Mannitol, a ROS scavenger, reversed the cytotoxic effect of ART and reduced DNA damage, and modulation of glutathione (GSH) levels was found to impact ROS and DNA damage induced by ART. Accumulation of ROS, increased DNA damage, and the resulting antiparasite effect suggest a causal relationship between ROS, DNA damage, and parasite death. Finally, we also show that ART-induced ROS production involves a potential role for NADPH oxidase, an enzyme involved in the production of superoxide anions. Our results with P. falciparum provide novel insights into previously unknown molecular mechanisms underlying the antimalarial activity of artemisinin derivatives and may help in the design of next-generation antimalarial drugs against the most virulent Plasmodium species.

  14. Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury

    PubMed Central

    Lin, Weiwei; Yuan, Na; Wang, Zhen; Cao, Yan; Fang, Yixuan; Li, Xin; Xu, Fei; Song, Lin; Wang, Jian; Zhang, Han; Yan, Lili; Xu, Li; Zhang, Xiaoying; Zhang, Suping; Wang, Jianrong

    2015-01-01

    Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis. PMID:26197097

  15. Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA.

    PubMed

    Speroni, Juliana; Federico, María Belén; Mansilla, Sabrina F; Soria, Gastón; Gottifredi, Vanesa

    2012-05-01

    The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling.