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Sample records for involves dna damage

  1. Involvement of DNA Damage Response Pathways in Hepatocellular Carcinoma

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    Sheau-Fang Yang

    2014-01-01

    Full Text Available 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.

  2. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    International Nuclear Information System (INIS)

    Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

    2012-01-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  3. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

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    Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

    2012-08-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  4. Role of DNA repair in repair of cytogenetic damages. Slowly repaired DNA injuries involved in cytogenetic damages repair

    International Nuclear Information System (INIS)

    Zaichkina, S.I.; Rozanova, O.M.; Aptikaev, G.F.; Ganassi, E.Eh.

    1989-01-01

    Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification

  5. Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response.

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    Fudrini Olivencia, Begonia; Müller, Andreas U; Roschitzki, Bernd; Burger, Sibylle; Weber-Ban, Eilika; Imkamp, Frank

    2017-10-25

    Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.

  6. Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue.

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    Hendershot, Jenna M; O'Brien, Patrick J

    2017-09-29

    DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr 162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1, N 6 -ethenoadenine (ϵA) lesion, and the rate of N -glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Involvement of DNA damage in hydroxyurea-mediated induction of endogenous murine retrovirus

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    Rascati, R.J.; Tennant, R.W.

    1979-04-30

    Hydroxyurea (HU) induces AKR cells to produce endogenous murine retrovirus at a low frequency (approx. 1 x 10/sup -5/), and DNA synthesis is required soon after treatment with HU for induction to be observed (i.e., no stable induction intermediate is formed). Induction by HU can be enhanced by simultaneous treatment with halogenated pyrimidines, with the concomitant appearance of a stable induction intermediate state. The effects of the two compounds are synergistic, indicating an actual stimulation of HU-mediated induction by iodode-oxyuridine. Since HU inhibits semiconservative replication, and since (/sup 3/H)bromodeoxyuridine is incorporated into the cellular genome predominantly by unscheduled DNA synthesis (repair replication) under these conditions, this stimulation appears to be the result of insertion into DNA of the thymidine analogs during the repair of HU-induced alterations in the DNA. The nature of HU damage to DNA is not defined; if single-strand breaks are involved, they may occur at a frequency <10/sup -8/ and escape detection, but induction could also be due to other alterations in DNA. The characteristics of induction by HU, therefore, are similar to those of induction by other DNA-damaging treatments such as ..gamma.. or x irradiation or methylcholanthrene. This suggests that these agents may induce by similar, if not identical, mechanisms. Further, the ability of halogenated pyrimidines to form a stable induction intermediate when incorporated by repair synthesis, similar to the intermediate formed when the analogs are incorporated during semiconservative replication, suggests that the same sites are involved for induction by damaging agents or by halogenated pyrimidine incorporation.

  8. Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

    International Nuclear Information System (INIS)

    Park, Iha; Avraham, Hava Karsenty

    2006-01-01

    Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving γ-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation

  9. Site-specific DNA damage at the GGG sequence by UVA involves acceleration of telomere shortening.

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    Oikawa, S; Tada-Oikawa, S; Kawanishi, S

    2001-04-17

    Telomere shortening is associated with cellular senescence. We investigated whether UVA, which contributes to photoaging, accelerates telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI-38 fibroblasts irradiated with UVA (365-nm light) decreased with increasing irradiation dose. Furthermore, UVA irradiation dose-dependently increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both WI-38 fibroblasts and HL-60 cells. To clarify the mechanism of the acceleration of telomere shortening, we investigated site-specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using (32)P 5'-end-labeled DNA fragments containing the telomeric oligonucleotide (TTAGGG)(4). UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5'-GGG-3' in telomere sequence. The amount of 8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)(7)CGC-3'] was approximately 5 times more than that in DNA fragment containing nontelomere sequence [5'-CGC(TGTGAG)(7)CGC-3']. Catalase did not inhibit this oxidative DNA damage, indicating no or little participation of H(2)O(2) in DNA damage. These results indicate that the photoexcited endogenous photosensitizer specifically oxidizes the central guanine of 5'-GGG-3' in telomere sequence to produce 8-oxodG probably through an electron-transfer reaction. It is concluded that the site-specific damage in telomere sequence induced by UVA irradiation may participate in the increase of telomere shortening rate.

  10. The role of hnRPUL1 involved in DNA damage response is related to PARP1.

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    Zehui Hong

    Full Text Available Heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1 -also known as adenovirus early region 1B-associated proteins 5 (E1B-AP5 - plays a role in RNA metabolism. Recently, hnRPUL1 has also been shown to be involved in DNA damage response, but the function of hnRPUL1 in response to DNA damage remains unclear. Here, we have demonstrated that hnRPUL1 is associated with PARP1 and recruited to DNA double-strand breaks (DSBs sites in a PARP1-mediated poly (ADP-ribosyl ation dependent manner. In turn, hnRPUL1 knockdown enhances the recruitment of PARP1 to DSBs sites. Specifically, we showed that hnRPUL1 is also implicated in the transcriptional regulation of PARP1 gene. Thus, we propose hnRPUL1 as a new component related to PARP1 in DNA damage response and repair.

  11. Kin3 protein, a NIMA-related kinase of Saccharomyces cerevisiae, is involved in DNA adduct damage response.

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    Moura, Dinara J; Castilhos, Bruna; Immich, Bruna F; Cañedo, Andrés D; Henriques, João A P; Lenz, Guido; Saffi, Jenifer

    2010-06-01

    Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.

  12. DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

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    Jatsenko, Tatjana; Sidorenko, Julia; Saumaa, Signe; Kivisaar, Maia

    2017-01-01

    Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.

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

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

    2016-01-01

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

  14. In situ action spectra suggest that DNA damage is involved in ultraviolet radiation-induced immunosuppression in humans

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    Hurks, H.M.H.; Out-Luiting, C.; Vermeer, B.-J.; Claas, F.H.J.; Mommaas, A.M. [University Hospital, Leiden (Netherlands)

    1997-07-01

    The mixed epidermal cell lymphocyte reaction (MECLR) is a commonly used method to study the immunomodulatory effects of UV radiation. The in vitro action spectrum for the MECLR showed that the UV-induced suppression of the MECLR responses is associated with UV-induced DNA damage. To investigate whether in vivo DNA damage also leads to the abrogation of the MECLR, in situ action spectra were made for the MECLR and the induction of thymine dimers (T<>T). Human skin, obtained from plastic surgery, was exposed to monochromatic light of 254, 297, 302 and 312nm. After irradiation, epidermal cells were isolated and used as stimulator cells in the MECLR or processed for flow cytometric detection of T<>T. On the basis of dose-response curves for each wavelength, the action spectyra for suppression of the MECLR and the induction of T<>T were calculated. These spectra showed close similarities, suggesting that, also in situ, UV-induced DNA damage is involved in the UV-induced suppression of the MECLR. Both action spectra showed a small decline from 254 nm to 302 nm, followed by a steep decline to 312 nm. These data show that, in situ, UVC can efficiently induce DNA damage and modulate cutaneous immune responses. (author).

  15. Structural biology of disease-associated repetitive DNA sequences and protein-DNA complexes involved in DNA damage and repair

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    Gupta, G.; Santhana Mariappan, S.V.; Chen, X.; Catasti, P.; Silks, L.A. III; Moyzis, R.K.; Bradbury, E.M.; Garcia, A.E.

    1997-07-01

    This project is aimed at formulating the sequence-structure-function correlations of various microsatellites in the human (and other eukaryotic) genomes. Here the authors have been able to develop and apply structure biology tools to understand the following: the molecular mechanism of length polymorphism microsatellites; the molecular mechanism by which the microsatellites in the noncoding regions alter the regulation of the associated gene; and finally, the molecular mechanism by which the expansion of these microsatellites impairs gene expression and causes the disease. Their multidisciplinary structural biology approach is quantitative and can be applied to all coding and noncoding DNA sequences associated with any gene. Both NIH and DOE are interested in developing quantitative tools for understanding the function of various human genes for prevention against diseases caused by genetic and environmental effects.

  16. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  17. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

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    Pinto-Fernandez, Adan; Kessler, Benedikt M

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  18. DUBbing cancer: Deubiquitylating enzymes involved in epigenetics, DNA damage and the cell cycle as therapeutic targets

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    Benedikt M Kessler

    2016-07-01

    Full Text Available Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs, have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  19. Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

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    Jean-Luc Perfettini

    Full Text Available DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM, which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env of human immunodeficiency virus-1 (HIV-1 in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein, as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

  20. Multiple and variable NHEJ-like genes are involved in resistance to DNA damage in Streptomyces ambofaciens

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    Grégory Hoff

    2016-11-01

    Full Text Available Non homologous end-joining (NHEJ is a double strand break (DSB repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the core NHEJ gene set constituted of conserved loci and the variable NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC 23877, not only the deletion of core genes but also that of variable genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

  1. Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays.

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    Vens, C.; Hofland, I.; Begg, A.C.

    2007-01-01

    Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta

  2. Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage.

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    Kanagaraj, Radhakrishnan; Parasuraman, Prasanna; Mihaljevic, Boris; van Loon, Barbara; Burdova, Kamila; König, Christiane; Furrer, Antonia; Bohr, Vilhelm A; Hübscher, Ulrich; Janscak, Pavel

    2012-09-01

    Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated by the MutY DNA glycosylase homologue (MUTYH). Here, by using formaldehyde crosslinking in mammalian cell extracts, we demonstrate that the WRN helicase/exonuclease defective in the premature aging disorder Werner syndrome (WS) is recruited to DNA duplex containing an 8-oxo-G:A mispair in a manner dependent on DNA polymerase λ (Polλ) that catalyzes accurate DNA synthesis over 8-oxo-G. Similarly, by immunofluorescence, we show that Polλ is required for accumulation of WRN at sites of 8-oxo-G lesions in human cells. Moreover, we show that nuclear focus formation of WRN and Polλ induced by oxidative stress is dependent on ongoing DNA replication and on the presence of MUTYH. Cell viability assays reveal that depletion of MUTYH suppresses the hypersensitivity of cells lacking WRN and/or Polλ to oxidative stress. Biochemical studies demonstrate that WRN binds to the catalytic domain of Polλ and specifically stimulates DNA gap filling by Polλ over 8-oxo-G followed by strand displacement synthesis. Our results suggest that WRN promotes long-patch DNA repair synthesis by Polλ during MUTYH-initiated repair of 8-oxo-G:A mispairs.

  3. Autophagy in DNA Damage Response

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    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  4. Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage

    Czech Academy of Sciences Publication Activity Database

    Kanagaraj, R.; Parasuraman, P.; Mihaljevic, B.; van Loon, B.; Burdová, Kamila; König, C.; Furrer, A.; Bohr, V.A.; Hübscher, U.; Janscak, P.

    2012-01-01

    Roč. 40, č. 17 (2012), s. 8449-8459 ISSN 0305-1048 Grant - others:Swiss National Science Foundation(CH) 31003A-129747/1; Swiss National Science Foundation(CH) 3100-109312/2; Oncosuisse(CH) KLS-02344-02-2009; NIH(US) Z01-AG000726-17 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : DNA repair * oxidative stress * MUTYH * WRN * Pol lambda Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

  5. Tet1-mediated DNA demethylation involves in neuron damage induced by bilirubin in vitro.

    Science.gov (United States)

    Gou, Panhong; Qi, Xiaoling; Yuan, Rui; Li, Haojie; Gao, Xiaoling; Wang, Junling; Zhang, Benzhong

    2018-01-01

    The aim of this study is to identify the role of Tet1-mediated DNA demethylation in the neurotoxicity caused by unconjugated bilirubin (UCB) in vitro. Primary neuronal cells after cultured for 72 h were exposed to UCB (0-100 μmol/L) for 24 h. Following exposure to UCB cytotoxicity was determined with the methyl tetrazolium (MTT) assay, reactive oxygen species (ROS) and caspase-3 activity in neuron cells were measured with the corresponding assay kits. The expression of Tet1 and Klotho was determined with RT-PCR at mRNA level and western blot at protein level. Our results showed that UCB can cause time-dependent and dose-dependent reduction of cell viability of neuronal cells, induce oxidative stress through increasing the production of ROS and increase caspase-3 activity. Quantitative real-time PCR and western blot analysis showed that UCB can inhibit Tet1 and Klotho expression in cultured neuronal cells at both the mRNA and protein level, respectively. These results are first to suggest UCB may, in part, exert its neurotoxicity through alteration of the neuronal antioxidant status and inhibition of Klotho and Tet1 gene expression. The elevation of DNA methylation in global genome through inhibition of Tet1 gene expression may, in part, play an important role in the neurotoxicity caused by UCB in vitro.

  6. Radiation damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, V.

    1978-01-01

    A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

  7. DNA Damage and Pulmonary Hypertension

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  9. DNA damage and carcinogenesis

    International Nuclear Information System (INIS)

    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 4 fold

  10. Endonuclease IV Is the Main Base Excision Repair Enzyme Involved in DNA Damage Induced by UVA Radiation and Stannous Chloride

    Directory of Open Access Journals (Sweden)

    Ellen S. Motta

    2010-01-01

    Full Text Available Stannous chloride (SnCl2 and UVA induce DNA lesions through ROS. The aim of this work was to study the toxicity induced by UVA preillumination, followed by SnCl2 treatment. E. coli BER mutants were used to identify genes which could play a role in DNA lesion repair generated by these agents. The survival assays showed (i The nfo mutant was the most sensitive to SnCl2; (ii lethal synergistic effect was observed after UVA pre-illumination, plus SnCl2 incubation, the nfo mutant being the most sensitive; (iii wild type and nfo mutants, transformed with pBW21 plasmid (nfo+ had their survival increased following treatments. The alkaline agarose gel electrophoresis assays pointed that (i UVA induced DNA breaks and fpg mutant was the most sensitive; (ii SnCl2-induced DNA strand breaks were higher than those from UVA and nfo mutant had the slowest repair kinetics; (iii UVA+SnCl2 promoted an increase in DNA breaks than SnCl2 and, again, nfo mutant displayed the slowest repair kinetics. In summary, Nfo protects E. coli cells against damage induced by SnCl2 and UVA+ SnCl2.

  11. DNA Damage, Repair, and Cancer Metabolism

    Science.gov (United States)

    Turgeon, Marc-Olivier; Perry, Nicholas J. S.; Poulogiannis, George

    2018-01-01

    Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors. PMID:29459886

  12. A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.

    LENUS (Irish Health Repository)

    2010-11-15

    DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1\\/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2\\/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2\\/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

  13. The cycad genotoxin MAM modulates brain cellular pathways involved in neurodegenerative disease and cancer in a DNA damage-linked manner.

    Science.gov (United States)

    Kisby, Glen E; Fry, Rebecca C; Lasarev, Michael R; Bammler, Theodor K; Beyer, Richard P; Churchwell, Mona; Doerge, Daniel R; Meira, Lisiane B; Palmer, Valerie S; Ramos-Crawford, Ana-Luiza; Ren, Xuefeng; Sullivan, Robert C; Kavanagh, Terrance J; Samson, Leona D; Zarbl, Helmut; Spencer, Peter S

    2011-01-01

    Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O⁶-methyldeoxyguanosine lesions, O⁶-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O⁶-mG DNA methyltransferase (MGMT) showed elevated O⁶-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer's disease.

  14. Radiation damage to DNA constituents

    International Nuclear Information System (INIS)

    Bergene, R.

    1977-01-01

    The molecular changes of the DNA molecule, in various systems exposed to inoizing radiation, have been the subject of a great number of studies. In the present work electron spin resonance spectroscopy (ESR) has been applied to irradiated crystalline systems, in particular single crystals of DNA subunits and their derivatives. The main conclusions about the molecular damage are based on this technique in combination with molecular orbital calculations. It should be emphasized that the ESR technique is restricted to damage containing unpaired electrons. These unstable intermediates called free radicals seem, however, to be involved in all molecular models describing the action of radiation on DNA. One of the premises for a detailed theory of the radiation induced reactions at the physico-chemical level seems to involve exact knowledge of the induced free radicals as well as the modes of their formation and fate. For DNA, as such, it is hardly possible to arrive at such a level of knowledge since the molecular complexity prevents selective studies of the many different radiation induced products. One possible approach is to study the free radicals formed in the constituents of DNA. In the present work three lines of approach should be mentioned. The first is based on the observation that radical formation in general causes only minor structural alterations to the molecule in question. The use of isotopes with different spin and magnetic moment (in particular deuterium) may also serve a source of information. Deuteration leads to a number of protons, mainly NH - and OH, becoming substituted, and if any of these are involved in interactions with unpaired protons the resonance pattern is influeneed. The third source of information is molecular orbital calculation. The electron spin density distribution is a function in the three dimensional space based on the system's electronic wave functions. This constitutes the basis for the idea that ESR data can be correlated with

  15. DNA Damage Induced Neuronal Death

    National Research Council Canada - National Science Library

    Kisby, Glen

    1999-01-01

    ... (nitrogen mustard or HN2) and the neurotoxic DNA-damaging agent methylazoxymethanol (MAM) using neuronal and astrocyte cell cultures from different brain regions of mice with perturbed DNA repair...

  16. Histone Modification Is Involved in Okadaic Acid (OA Induced DNA Damage Response and G2-M Transition Arrest in Maize.

    Directory of Open Access Journals (Sweden)

    Hao Zhang

    Full Text Available Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA, a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph signals was altered under OA treatment. Reactive oxygen species (ROS was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

  17. DNA damage in neurodegenerative diseases

    International Nuclear Information System (INIS)

    Coppedè, Fabio; Migliore, Lucia

    2015-01-01

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: 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

  18. DNA damage in neurodegenerative diseases

    Energy Technology Data Exchange (ETDEWEB)

    Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

    2015-06-15

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: 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

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Identification of intermediate-size non-coding RNAs involved in the UV-induced DNA damage response in C. elegans.

    Directory of Open Access Journals (Sweden)

    Aqian Li

    Full Text Available BACKGROUND: A network of DNA damage response (DDR mechanisms functions coordinately to maintain genome integrity and prevent disease. The Nucleotide Excision Repair (NER pathway is known to function in the response to UV-induced DNA damage. Although numbers of coding genes and miRNAs have been identified and reported to participate in UV-induced DNA damage response (UV-DDR, the precise role of non-coding RNAs (ncRNAs in UV-DDR remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used high-throughput RNA-sequencing (RNA-Seq to discover intermediate-size (70-500 nt ncRNAs (is-ncRNAs in C. elegans, using the strains of L4 larvae of wild-type (N2, UV-irradiated (N2/UV100 and NER-deficient mutant (xpa-1, and 450 novel non-coding transcripts were initially identified. A customized microarray assay was then applied to examine the expression profiles of both novel transcripts and known is-ncRNAs, and 57 UV-DDR-related is-ncRNA candidates showed expression variations at different levels between UV irradiated strains and non- irradiated strains. The top ranked is-ncRNA candidates with expression differences were further validated by qRT-PCR analysis, of them, 8 novel is-ncRNAs were significantly up-regulated after UV irradiation. Knockdown of two novel is-ncRNAs, ncRNA317 and ncRNA415, by RNA interference, resulted in higher UV sensitivity and significantly decreased expression of NER-related genes in C. elegans. CONCLUSIONS/SIGNIFICANCE: The discovery of above two novel is-ncRNAs in this study indicated the functional roles of is-ncRNAs in the regulation of UV-DDR network, and aided our understanding of the significance of ncRNA involvement in the UV-induced DNA damage response.

  1. The DNA damage response during mitosis

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  2. The DNA damage response during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van, E-mail: m.vugt@umcg.nl

    2013-10-15

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

  3. The DNA damage response during mitosis.

    Science.gov (United States)

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

    2013-10-01

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

  4. Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

    Science.gov (United States)

    Redrejo-Rodríguez, Modesto; Vigouroux, Armelle; Mursalimov, Aibek; Grin, Inga; Alili, Doria; Koshenov, Zhanat; Akishev, Zhiger; Maksimenko, Andrei; Bissenbaev, Amangeldy K; Matkarimov, Bakhyt T; Saparbaev, Murat; Ishchenko, Alexander A; Moréra, Solange

    2016-01-01

    Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Opposed arsenite-mediated regulation of p53-survivin is involved in neoplastic transformation, DNA damage, or apoptosis in human keratinocytes

    International Nuclear Information System (INIS)

    Li, Yuan; Jiang, Rongrong; Zhao, Yue; Xu, Yuan; Ling, Min; Pang, Ying; Shen, Lu; Zhou, Yun; Zhang, Jianping; Zhou, Jianwei; Wang, Xinru; Liu, Qizhan

    2012-01-01

    Highlights: ► Different concentrations of arsenite cause biphasic effects in HaCaT cells. ► p53-survivin signal pathway plays a role in arsenite-induced biphasic effects. ► ERKs inactivate p53, but improve survivin expression by NF-κB/mot-2. ► JNKs block survivin expression by preventing p53 from mdm2-mediated degradation. ► ERKs and JNKs play roles in arsenite-induced biphasic effects. -- Abstract: Biphasic dose–response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose–response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. Our present study shows that, for human keratinocytes (HaCaT) cells, a low concentration of arsenite activates extracellular signal-regulated kinases (ERKs), which leads to up-regulation of nuclear factor κB (NF-κB) binding to DNA and to elevated, NF-κB-dependent expression of mot-2 (a p53 inhibitor) and survivin (an inhibitor of apoptosis). Activation of p53 is blocked, and neoplastic transformation is enhanced. Inhibition of ERKs reduces cell proliferation and neoplastic transformation. In contrast, a high concentration of arsenite activates c-Jun N-terminal kinases (JNKs), positive regulators of p53, by binding to p53 and preventing its murine double minute 2 (mdm2)-mediated degradation. The elevated levels of p53 lead to repair of DNA damage and apoptosis. Inhibition of JNKs increases DNA damage but decreases apoptosis. By identifying a mechanism whereby ERKs and JNKs-mediated regulation of the p53-survivin signal pathway is involved in the biphasic effects of arsenite on human keratinocytes, our data expand understanding of arsenite-induced cell proliferation, neoplastic transformation, DNA damage, and apoptosis.

  6. DNA damage by Auger emitters

    International Nuclear Information System (INIS)

    Martin, R.F.; d'Cunha, Glenn; Gibbs, Richard; Murray, Vincent; Pardee, Marshall; Allen, B.J.

    1988-01-01

    125 I atoms can be introduced at specific locations along a defined DNA target molecule, either by site-directed incorporation of an 125 I-labelled deoxynucleotide or by binding of an 125 I-labelled sequence-selective DNA ligand. After allowing accumulation of 125 I decay-induced damage to the DNA, application of DNA sequencing techniques enables positions of strand breaks to be located relative to the site of decay, at a resolution corresponding to the distance between adjacent nucleotides [0.34 nm]. Thus, DNA provides a molecular framework to analyse the extent of damage following [averaged] individual decay events. Results can be compared with energy deposition data generated by computer-simulation methods developed by Charlton et al. The DNA sequencing technique also provides information about the chemical nature of the termini of the DNA chains produced following Auger decay-induced damage. In addition to reviewing the application of this approach to the analysis of 125 I decay induced DNA damage, some more recent results obtained by using 67 Ga are also presented. (author)

  7. The essential DNA polymerases δ and ε are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Halas, A.; Policinska, Z.; Baranowska, H.; Jachymczyk, W.J.

    1999-01-01

    We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases δ and ε showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases δ and ε are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Heidor, Renato [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Furtado, Kelly Silva; Ortega, Juliana Festa [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Oliveira, Tiago Franco de [Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Tavares, Paulo Eduardo Latorre Martins; Vieira, Alessandra; Miranda, Mayara Lilian Paulino [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Purgatto, Eduardo [Laboratory of Food Chemistry and Biochemistry, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Moreno, Fernando Salvador, E-mail: rmoreno@usp.br [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil)

    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.

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

    International Nuclear Information System (INIS)

    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

    2014-01-01

    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

  10. (UVB)-induced DNA damage

    African Journals Online (AJOL)

    Jane

    2011-08-17

    Aug 17, 2011 ... E-mail: renu2498@hotmail.com. Abbreviations: POE, Pandanus ordoratissimus extract; KSCs, keratinocyte stem cells; AAG, ascorbyl glucoside. as the major cause of human skin cancer. It is well established that UVB induced DNA damage by photoi- somerization, resulting in the formation of the 6-4 photo-.

  11. OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

    Science.gov (United States)

    Liu, Zhong-Jie; Zhao, Wei; Zhang, Qing-Guo; Li, Le; Lai, Lu-Ying; Jiang, Shan; Xu, Shi-Yuan

    2015-01-01

    Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1) which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS). SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use. PMID:26161242

  12. DNA damage and repair mechanism. [DNA damage and repair mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, L.

    1976-01-01

    The ability of cells to survive in an environment specifically damaging to its DNA can be attributed to a variety of inherent repair mechanisms. This is a form of repair in which alterations are directly reversed to their original form. This reversibility is exemplified by the photoreactivation of ultraviolet-induced pyrimidine dimers. This phenomenon is attributable to the action of an enzyme, photolyase (photoreactivating enzyme), which is able to monomerize the uv-induced pyrimidine dimers in the presence of 320 to 370 nm light. Dilution of damage can be effected through a series of sister chromatid exchanges, controlled by recombinational mechanisms as a postreplication event. In this form of repair, replication proceeds to the point of damage, stops and resumes at the point of the next initiation site resulting in a gap in the newly synthesized daughter strand. It is presumed that those strands containing damaged regions exchange with undamaged regions of other DNA, strands, resulting in the eventual dilution of such damage.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nagane, Masaki; Yasui, Hironobu; Sakai, Yuri; Yamamori, Tohru [Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Niwa, Koichi [Laboratory of Biochemistry, Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri 099-2493 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Inanami, Osamu, E-mail: inanami@vetmed.hokudai.ac.jp [Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan)

    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.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Evaluation of the DNA damaging effects of amitraz on human ...

    Indian Academy of Sciences (India)

    mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following ...

  16. Sensitization of Tumor to {sup 212}Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

    Energy Technology Data Exchange (ETDEWEB)

    Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E. [Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Brechbiel, Martin W., E-mail: martinwb@mail.nih.gov [Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)

    2013-03-15

    Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using {sup 212}Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by {sup 212}Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with {sup 212}Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. {sup 212}Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling.

  17. Sensitization of Tumor to 212Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

    International Nuclear Information System (INIS)

    Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E.; Brechbiel, Martin W.

    2013-01-01

    Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using 212 Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by 212 Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with 212 Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. 212 Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling

  18. DNA Damage in Plant Herbarium Tissue

    OpenAIRE

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

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the ac...

  19. The DNA damage response during mitosis

    NARCIS (Netherlands)

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

    2013-01-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance

  20. Radiation, DNA damage and cancer

    International Nuclear Information System (INIS)

    Hall, J.; Angele, S.

    1999-01-01

    The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (Atm) has recently been shown to be a tumour suppressor gene in T-cell pro lymphocytic leukaemia, and there is increasing evidence that individuals with one mutated Atm or Nijmegen breakage syndrome (Nbs) allele have an increased predisposition to cancer. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  1. Radiation, DNA damage and cancer

    Energy Technology Data Exchange (ETDEWEB)

    Hall, J.; Angele, S. [Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon (France)

    1999-04-01

    The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (Atm) has recently been shown to be a tumour suppressor gene in T-cell pro lymphocytic leukaemia, and there is increasing evidence that individuals with one mutated Atm or Nijmegen breakage syndrome (Nbs) allele have an increased predisposition to cancer. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  2. Bromodomain proteins: repairing DNA damage within chromatin.

    Science.gov (United States)

    Chiu, Li-Ya; Gong, Fade; Miller, Kyle M

    2017-10-05

    Genome surveillance and repair, termed the DNA damage response (DDR), functions within chromatin. Chromatin-based DDR mechanisms sustain genome and epigenome integrity, defects that can disrupt cellular homeostasis and contribute to human diseases. An important chromatin DDR pathway is acetylation signalling which is controlled by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes, which regulate acetylated lysines within proteins. Acetylated proteins, including histones, can modulate chromatin structure and provide molecular signals that are bound by acetyl-lysine binders, including bromodomain (BRD) proteins. Acetylation signalling regulates several DDR pathways, as exemplified by the preponderance of HATs, HDACs and BRD proteins that localize at DNA breaks to modify chromatin for lesion repair. Here, we explore the involvement of acetylation signalling in the DDR, focusing on the involvement of BRD proteins in promoting chromatin remodelling to repair DNA double-strand breaks. BRD proteins have widespread DDR functions including chromatin remodelling, chromatin modification and transcriptional regulation. We discuss mechanistically how BRD proteins read acetylation signals within chromatin to trigger DDR and chromatin activities to facilitate genome-epigenome maintenance. Thus, DDR pathways involving BRD proteins represent key participants in pathways that preserve genome-epigenome integrity to safeguard normal genome and cellular functions.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  3. DNA damage in plant herbarium tissue.

    NARCIS (Netherlands)

    Staats, M.; Cuenca, A.; Richardson, J.E.; Ginkel, R.V.; Petersen, G.; Seberg, O.; Bakker, F.T.

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of

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

    Science.gov (United States)

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

    2017-10-27

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

  5. Detection of DNA damage by comet fluorescence in situ hybridization.

    Science.gov (United States)

    Schlörmann, Wiebke; Glei, Michael

    2012-01-01

    Comet fluorescence in situ hybridization (Comet-FISH) is a useful method to detect overall and region-specific DNA damage in individual cells. Two well-established methods are combined, the Comet assay (single cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay is the method of choice for the detection of DNA damage. With the alkaline version the influence of specific substances such as water pollutants or ingredients of food on individual cells can be easily measured. The Comet assay involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. Damaged DNA migrates from the nucleus (head of the comet) forming a tail. The percentage of DNA in the tail correlates with the degree of DNA strand breaks (DNA damage). The combination of FISH with the Comet assay uses labeled probes which hybridize specifically to selected DNA sequences. This allows the detection of specific DNA damage or repair capacity in single cells. Here we present exemplarily the Comet-FISH method by detection of DNA damage using hydrogen peroxide as a genotoxic model substrate.

  6. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1985-07-01

    Radiation damage to DNA results from the direct interaction of radiation with DNA where positive ions, electrons and excited states are formed in the DNA, and the indirect effect where radical species formed in the surrounding medium by the radiation attack the DNA. The primary mechanism proposed for radiation damage, by the direct effect, is that positive and negative ions formed within the DNA strand migrate through the stacked DNA bases. The ions can then recombine, react with the DNA bases most likely to react by protonation of the anion and deprotonation or hydroxylation of the cation or transfer out of the DNA chain to the surrounding histone protein. This work as aimed at understanding the possible reactions of the DNA base ion radicals, as well as their initial distribution in the DNA strand. 31 refs

  7. uv photobiology: DNA damage and repair

    International Nuclear Information System (INIS)

    Sutherland, B.M.

    1978-01-01

    The following topics are discussed: targets that determine the fate of the cell when uv light interacts with a cell; comparison of action spectrum for a given biological effect with the absorption spectrum of different biological macromolecules; biological effects of damage to DNA; measurement of mutations; chemical damage to DNA; photoreactivation; role of pyrimidine dimers in induction of skin cancer by uv

  8. Cellular responses to environmental DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Inflammation, oxidative DNA damage, and carcinogenesis

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  10. DNA Damage in Plant Herbarium Tissue

    Science.gov (United States)

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

    2011-01-01

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

  11. DNA damage in plant herbarium tissue.

    Science.gov (United States)

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

    2011-01-01

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

  12. DNA Damage Signals and Space Radiation Risk

    Science.gov (United States)

    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.

  13. Repair of DNA damage in Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Evans, D.M.

    1984-01-01

    The repair of DNA lesions in Deinococcus radiodurans was examined with particular reference to DNA excision repair of ultraviolet light (UV) induced pyrimidine dimers. The characteristics of excision repair via UV endonucleases α and β in vivo varied with respect to (a) the substrate range of the enzymes, (b) the rate of repair of DNA damage (c) the requirement for a protein synthesised in response to DNA damage to attenuate exonuclease action at repairing regions. UV endonuclease α is postulated to incise DNA in a different manner from UV endonuclease β thus defining the method of subsequent repair. Several DNA damage specific endonuclease activities independent of α and β are described. Mutations of the uvsA, uvsF and uvsG genes resulted in an increase in single-strand breaks in response to DNA damage producing uncontrolled DNA degradation. Evidence is presented that these genes have a role in limiting the access of UV endonuclease β to DNA lesions. uvsF and uvsG are also shown to be linked to the mtoA gene. Mutation of uvsH and reo-1 produces further distinct phenotypes which are discussed. An overall model of excision repair of DNA damage in Deinococcus radiodurans is presented. (author)

  14. Stress-induced DNA Damage biomarkers: Applications and limitations

    Science.gov (United States)

    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.

  15. Processing of free radical damaged DNA bases

    International Nuclear Information System (INIS)

    Wallace, S.

    2003-01-01

    Free radicals produced during the radiolysis of water gives rise to a plethora of DNA damages including single strand breaks, sites of base loss and a wide variety of purine and pyrimidine base lesions. All these damages are processed in cells by base excision repair. The oxidative DNA glycosylases which catalyze the first step in the removal of a base damage during base excision repair evolved primarily to protect the cells from the deleterious mutagenic effects of single free radical-induced DNA lesions arising during oxidative metabolism. This is evidenced by the high spontaneous mutation rate in bacterial mutants lacking the oxidative DNA glycosylases. However, when a low LET photon transverses the DNA molecule, a burst of free radicals is produced during the radiolysis of water that leads to the formation of clustered damages in the DNA molecule, that are recognized by the oxidative DNA glycosylases. When substrates containing two closely opposed sugar damages or base and sugar damages are incubated with the oxidative DNA glycosylases in vitro, one strand is readily incised by the lyase activity of the DNA glycosylase. Whether or not the second strand is incised depends on the distance between the strand break resulting from the incised first strand and the remaining DNA lesion on the other strand. If the lesions are more than two or three base pairs apart, the second strand is readily cleaved by the DNA glycosylase, giving rise to a double strand break. Even if the entire base excision repair system is reconstituted in vitro, whether or not a double strand break ensues depends solely upon the ability of the DNA glycosylase to cleave the second strand. These data predicted that cells deficient in the oxidative DNA glycosylases would be radioresistant while those that overproduce an oxidative DNA glycosylase would be radiosensitive. This prediction was indeed borne in Escherichia coli that is, mutants lacking the oxidative DNA glycosylases are radioresistant

  16. Experimental study of oxidative DNA damage

    DEFF Research Database (Denmark)

    Loft, S; Deng, Xiaohong; Tuo, J

    1998-01-01

    of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran...... studies provide powerful tools to investigate agents inducing and preventing oxidative damage to DNA and its role in carcinogenesis. So far, most animal experiments have concerned 8-oxodG and determination of additional damaged bases should be employed. An ideal animal model for prevention of oxidative......Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical...

  17. Electrochemical DNA Sensors for Detection of DNA Damage

    Directory of Open Access Journals (Sweden)

    Ana Maria Oliveira Brett

    2005-11-01

    Full Text Available Electrochemical devices have received particular attention due to their rapiddetection and great sensitivity for the evaluation of DNA-hazard compounds interactionmechanisms. Several types of bioanalytical method use nucleic acids probes to detect DNAdamage. This article reviews current directions and strategies in the development andapplications of electrochemical DNA sensors for the detection of DNA damage.

  18. Imaging the DNA damage response with PET and SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Knight, James C.; Koustoulidou, Sofia; Cornelissen, Bart [University of Oxford, CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, Oxford (United Kingdom)

    2017-06-15

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

  19. DNA damage induced by radionuclide internal irradiation

    International Nuclear Information System (INIS)

    Cui Fengmei; Zhao Jingyong; Hong Chengjiao; Lao Qinhua; Wang Liuyi; Yang Shuqin

    2004-01-01

    Objective: To study the DNA damage of peripheral blood mononuclear cell (PBMC) in rats exposed to radionuclide internal irradiation. Methods: The radionuclides were injected into the rats and single cell get electrophoresis (SCGE) was performed to detect the length of DNA migration in the rat PBMC. Results: DNA migration in the rat PBMC increased with accumulative dose or dose-rate. It showed good relationship of dose vs. response and of dose-rate vs. response, both relationship could be described as linear models. Conclusion: Radionuclide internal irradiation could cause DNA damage in rat PBMC. (authors)

  20. Visualization of complex DNA damage along accelerated ions tracks

    Science.gov (United States)

    Kulikova, Elena; Boreyko, Alla; Bulanova, Tatiana; Ježková, Lucie; Zadneprianetc, Mariia; Smirnova, Elena

    2018-04-01

    The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

  1. Early models of DNA damage formation

    International Nuclear Information System (INIS)

    Śmiałek, Małgorzata A

    2012-01-01

    Quantification of DNA damage, induced by various types of incident radiation as well as chemical agents, has been the subject of many theoretical and experimental studies, supporting the development of modern cancer therapy. The primary observations showed that many factors can lead to damage of DNA molecules. It became clear that the development of experimental techniques for exploring this phenomenon is required. Another problem was simultaneously dealt with, anticipating on how the damage is distributed within the double helix of the DNA molecule and how the single strand break formation and accumulation can influence the lethal double strand break formation. In this work the most important probabilistic models for DNA strand breakage and damage propagation are summarized and compared.

  2. DNA damage in the oocytes SACs

    Czech Academy of Sciences Publication Activity Database

    Macůrek, Libor

    2016-01-01

    Roč. 15, č. 4 (2016), s. 491-492 ISSN 1538-4101 Institutional support: RVO:68378050 Keywords : DNA damage response * oocyte * meiosis * checkpoint Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.530, year: 2016

  3. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1993-12-01

    In this project the author has proposed several mechanisms for radiation damage to DNA and its constituents, and has detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work he has completed several experiments on the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges and their reactions on DNA, investigated protonation reactions in DNA base anions, and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics evolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this years work to a consideration of ionization energies of various components of the DNA deoxyribose backbone and resulting neutral sugar radicals. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, four are in press, one is submitted and several more are in preparation. Four papers have been presented at scientific meetings. This years effort will include another review article on the open-quotes Electron Spin Resonance of Radiation Damage to DNAclose quotes

  4. Somatic DNA Damages in Cardiovascular Autonomic Neuropathy

    OpenAIRE

    Supriya Simon, A.; Dinesh Roy, D.; Jayapal, V.; Vijayakumar, T.

    2010-01-01

    Cardiovascular autonomic neuropathy (CAN) is one of the most clinically significant complications of diabetes mellitus. Even though many ethological factors have been attributed for the pathogenesis of this disease no attempts were made to correlate DNA damage as a causative factor. Hence the present study was undertaken to asses the extent of somatic DNA damages by cytokinesis-block micronuclei assay (CBMN). An attempt is also being made to correlate the habits and/or risk factors and socioe...

  5. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2016-02-01

    Full Text Available 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

  7. DNA damage assessment by visualization and quantification of DNA damage response

    International Nuclear Information System (INIS)

    Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

    2017-01-01

    DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

  8. Influence of diet on oxidative DNA damage, uracil misincorporation and DNA repair capability.

    Science.gov (United States)

    Prado, Renato Paschoal; dos Santos, Bruna Fornazari; Pinto, Carla Lombardi de Souza; de Assis, Kátia Regina Carvalho; Salvadori, Daisy Maria Fávero; Ladeira, Marcelo Sady Plácido

    2010-09-01

    The contribution of diet to cancer ranges from 10 to 80%. The low ingestion of antioxidants and enzymatic cofactors involved in DNA repair and methylation reactions and the high ingestion of chemical additives present in the modern diet, associated with genetic factors, could lead to genomic instability and the hypomethylation of proto-oncogenes, thus contributing to development of genetic-related diseases such as cancer. The present study evaluated the influence of diet on the level of oxidative DNA damage, misincorporated uracil and DNA repair capability in peripheral blood lymphocytes from two groups of individuals with antagonist diets as follows: (i) 49 healthy individuals with a diet rich in organic products, whole grains, fruit and vegetables and poor in processed foods (Group I) and (ii) 56 healthy individuals with diet rich in processed foods and poor in fruit and vegetables (Group II). Oxidative DNA damage, uracil incorporation and DNA repair capability were assessed by the comet assay. The individuals in Group I presented lower levels of oxidative DNA damage (oxidized purines and pyrimidines) and lower levels of DNA damage induced by ex vivo treatment with hydrogen peroxide (H(2)O(2)) than those individuals in Group II. The analysis of our results suggests that a diet rich in organic products, integral grains, fruit and vegetables and poor in industrialized products can protect against oxidative DNA damage and DNA damage induced by H(2)O(2).

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

    Science.gov (United States)

    Boiteux, Serge; Jinks-Robertson, Sue

    2013-01-01

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

  10. DNA Damage Triggers Golgi Dispersal via DNA-PK and GOLPH3

    OpenAIRE

    Farber-Katz, Suzette E.; Dippold, Holly C.; Buschman, Matthew D.; Peterman, Marshall C.; Xing, Mengke; Noakes, Christopher J.; Tat, John; Ng, Michelle M.; Rahajeng, Juliati; Cowan, David M.; Fuchs, Greg J.; Zhou, Huilin; Field, Seth J.

    2014-01-01

    The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA...

  11. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  12. Molecular models for DNA damaged by photoreaction

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  13. Delayed chromosomal instability induced by DNA damage

    International Nuclear Information System (INIS)

    Morgan, W.F.; Marder, B.A.; Day, J.P.

    1994-01-01

    Cellular exposure to DNA damaging agents rapidly results in a dose dependent increase in chromosomal breakage and gross structural chromosomal rearrangements. Over recent years, evidence has been accumulating indicating genomic instability can manifest multiple generations after cellular exposure to physical and chemical DNA damaging agents. Genomic instability manifests in the progeny of surviving cells, and has been implicated in mutation, gene application, cellular transformation, and cell killing. To investigate chromosome instability following DNA damage, we have used fluorescence in situ hybridization to detect chromosomal rearrangements in a human/hamster somatic hybrid cell line following exposure to ionizing radiation. Delayed chromosomal instability was detected when multiple populations of uniquely arranged metaphases were observed in clonal isolates raised from single cells surviving X-irradiation many generations after exposure. At higher radiation doses, chromosomal instability was observed in a relatively high frequency of surviving clones and, in general, those clones showed delayed chromosome instability also showed reduced survival as measured by colony forming ability

  14. Radiation damage of DNA. Model for direct ionization of DNA

    International Nuclear Information System (INIS)

    Kobayashi, Kazuo; Tagawa, Seiichi

    2004-01-01

    Current aspects of radiation damage of DNA, particularly induced by the direct effect of radiation, and author's method of pulse radiolysis are described in relation to behavior of ions formed by radiation and active principles to induce the strand break. In irradiation of DNA solution in water, the direct effect of radiation is derived from ionization of DNA itself and indirect one, from the reaction between DNA and radicals generated from water molecules and the former direct one has been scarcely investigated due to difficulty of experimental approach. Radicals generated in sugar moiety of DNA are shown important in the strand break by recent studies on crystalline DNA irradiated by X-ray, DNA solution by electron and photon beams, hydrated DNA by γ-ray and by high linear energy transfer (LET) ion. Author's pulse radiolysis studies have revealed behaviors of guanine and adenine radical cations in dynamics of DNA oxidation. Since reactions described are the model, the experimental approach is thought necessary for elucidation of the actually occurring DNA damage in living cells. (N.I.)

  15. DNA damage checkpoint and repair centers

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2004-01-01

    recognition and binding of DNA ends by the Mre11 complex and Ku70/80; second, end-processing and binding of single-stranded DNA by replication protein A, which recruits checkpoint proteins; third, recombinational repair during S and G(2) phase; and fourth, disassembly of foci and resumption of the cell cycle.......In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into focal assemblies. These foci are highly dynamic giga-dalton structures capable of simultaneously repairing multiple DNA lesions. Moreover, the composition of these repair centers depends...... on the nature of the DNA lesion and is tightly coordinated with progression of the cell cycle. Components of DNA repair centers are regulated by post-translational modifications such as phosphorylation, ubiquitination and sumoylation. Repair foci progress through four distinct stages: first, DNA damage...

  16. Dietary modulation of DNA damage in human.

    Science.gov (United States)

    Simic, M G; Bergtold, D S

    1991-01-01

    Manipulation of human diet can modulate urinary biomarkers of oxidative DNA base damage (UBODBD), reflecting changes in levels of DNA damage. When dietary composition is maintained but caloric intake is decreased (caloric restriction), UBODBD excretion is suppressed. At isocaloric dietary intake the level of damage depends on diet composition. For diets consisting of foods containing carbohydrates, proteins, and fats but lacking fruits and vegetables, the level of damage is higher than for diets including fruits and vegetables, which are rich in natural antioxidants. Assay of urinary biomarkers is suggested as a potential test for quantitative assessment of the carcinogenic or anticarcinogenic properties of foods, food components, and diets and for individual responses to nutritional regimens.

  17. Vitamin C for DNA damage prevention

    Energy Technology Data Exchange (ETDEWEB)

    Sram, Radim J., E-mail: sram@biomed.cas.cz [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic); Binkova, Blanka; Rossner, Pavel [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic)

    2012-05-01

    The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2 Prime -deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 {mu}mol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with {gamma}-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 {mu}mol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 {mu}mol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

  18. Vitamin C for DNA damage prevention

    International Nuclear Information System (INIS)

    Sram, Radim J.; Binkova, Blanka; Rossner, Pavel

    2012-01-01

    The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

  19. DNA damage response during mouse oocyte maturation

    Czech Academy of Sciences Publication Activity Database

    Mayer, Alexandra; Baran, Vladimír; Sakakibara, Y.; Brzáková, Adéla; Ferencová, Ivana; Motlík, Jan; Kitajima, T.; Schultz, R. M.; Šolc, Petr

    2016-01-01

    Roč. 15, č. 4 (2016), s. 546-558 ISSN 1538-4101 R&D Projects: GA MŠk LH12057; GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : double strand DNA breaks * DNA damage * MRE11 * meiotic maturation * mouse oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.530, year: 2016

  20. Oxidation of DNA: damage to nucleobases.

    Science.gov (United States)

    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

  1. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1987-01-01

    Several mechanisms are proposed for radiation damage to DNA and its constituents, and a series of experiments utilizing electron spin resonance spectrometry have been used to test the proposed mechanisms. In the past we have concentrated chiefly on investigating irradiated systems of DNA constituents. In this year's effort we have concentrated on radiation effects on DNA itself. In addition studies of radiation effects on lipids and model compounds have been performed which shed light on the only other proposed site for cell kill, the membrane

  2. Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Blasiak, Janusz; Arabski, Michal; Krupa, Renata; Wozniak, Katarzyna; Rykala, Jan; Kolacinska, Agnieszka; Morawiec, Zbigniew; Drzewoski, Jozef; Zadrozny, Marek

    2004-10-04

    Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results

  3. Vascular Aging from DNA Damage to Protection

    NARCIS (Netherlands)

    M. Durik (Matej)

    2012-01-01

    textabstractAging is highly associated with development of cardiovascular disease; however, the underlying mechanisms of these processes are not well understood. Recent advancements in aging research underscore the importance of DNA damage and subsequent cellular senescence in the aging process. In

  4. DNA damage response: multilevel proteomics gains momentum.

    Science.gov (United States)

    Daub, Henrik

    2012-04-27

    In this issue of Molecular Cell, Beli et al. (2012) introduce a multilevel proteomics approach for parallel quantification of protein phosphorylation, acetylation, and abundance and apply this to the complex signaling network of the DNA damage response. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    Science.gov (United States)

    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. Repair of damaged DNA in vivo: Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Hanawalt, P.C.

    1987-09-01

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

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

    International Nuclear Information System (INIS)

    Hanawalt, P.C.

    1987-09-01

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

  8. mRNA Processing Factor CstF-50 and Ubiquitin Escort Factor p97 Are BRCA1/BARD1 Cofactors Involved in Chromatin Remodeling during the DNA Damage Response.

    Science.gov (United States)

    Fonseca, Danae; Baquero, Jorge; Murphy, Michael R; Aruggoda, Gamage; Varriano, Sophia; Sapienza, Carmen; Mashadova, Oksana; Rahman, Shadaqur; Kleiman, Frida E

    2018-02-15

    The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR. Copyright © 2018 Fonseca et al.

  9. DNA damage checkpoint recovery and cancer development

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Haiyong [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Zhang, Xiaoshan [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States); Teng, Lisong, E-mail: lsteng@zju.edu.cn [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Legerski, Randy J., E-mail: rlegersk@mdanderson.org [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States)

    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

  10. Does radial nuclear organisation influence DNA damage?

    Science.gov (United States)

    Gazave, Elodie; Gautier, Philippe; Gilchrist, Susan; Bickmore, Wendy A

    2005-01-01

    It has been suggested that chromatin at the nuclear periphery could act to shield DNA sequences in the nuclear interior from damage. To test this hypothesis, we have examined the nuclear distribution of sites of DNA repair induced by oxidation or UV-C. We do not detect more damage (repair) at the nuclear periphery than in the nuclear interior. In fact, contrary to the body guard hypothesis, there is an excess of damage detected in the nuclear interior. This is further supported by sequence comparison between genes on human chromosomes 18 or 19, and their counterparts in the chimpanzee. The synonymous substitution rate for genes on chromosome 19, which is located towards the centre of the human nucleus, was higher than that for genes on chromosome 18, which is located at the nuclear periphery. We conclude that chromatin at the periphery of the human nucleus is not able to protect more internally located sequences from damage and mutation. We suggest that features of the chromatin structure, or base composition, of sequences in the nuclear centre make them more susceptible to damage.

  11. DNA damaging potential of Ganoderma lucidum extracts.

    Science.gov (United States)

    Gurovic, María Soledad Vela; Viceconte, Fátima R; Pereyra, Marcelo T; Bidegain, Maximiliano A; Cubitto, María Amelia

    2018-05-10

    Ganoderma lucidum (Lingzhi or Reishi) is a medicinal mushroom historically used in Asian countries to treat a wide variety of diseases and prolong life. In the last years, G. lucidum has been internationally recognized as an effective adjuvant in cancer treatment. Among active components, the most recent research indicates that polysaccharides modulate the immune response favoring the recovery from toxicity of chemo and radiotherapy while triterpenes are cytotoxic to tumoral cells mainly by altering gene expression. Beyond this body of evidence on the efficacy of G. lucidum in cancer treatment, it is not yet understood whether these extracts exert the same mechanisms of action than current antitumoral drugs. In this study, we tested the DNA damaging potential of G. lucidum extracts by the β-galactosidase biochemical prophage induction assay (BIA) using doxorubicin, a DNA intercalating agent, as a positive control. This assay was traditionally used to screen microbial metabolites towards antitumoral agents. Here, we used this bacterial assay for the first time to assess DNA damage of herbal drugs. After a bioguided assay, only a purified fraction of G. lucidum containing a mixture of C16 and C18:1 fatty acids exerted weak activity which could not be attributed to direct interaction with DNA. At the same concentrations, the induction observed for doxorubicin was clearly contrasting. The micro BIA assay could be successfully used to demonstrate differences in cellular effects between G. lucidum extracts and doxorubicin. These results showed that G. lucidum extracts display weak DNA damaging potential. Since DNA injury promotes aging and cancer, our results substantiate the traditional use of this mushroom to prolong life. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Short communication Sperm DNA damage in relation to lipid ...

    African Journals Online (AJOL)

    Leyland Fraser

    2017-03-08

    LPO) and sperm DNA damage following freezing-thawing of boar semen in different extenders. The comet assay was used to measure the extent of sperm DNA damage in a cryoprotectant-free extender or in cryoprotectant-based ...

  13. Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage

    Directory of Open Access Journals (Sweden)

    Robert L. Eoff

    2010-01-01

    Full Text Available DNA is a chemically reactive molecule that is subject to many different covalent modifications from sources that are both endogenous and exogenous in origin. The inherent instability of DNA is a major obstacle to genomic maintenance and contributes in varying degrees to cellular dysfunction and disease in multi-cellular organisms. Investigations into the chemical and biological aspects of DNA damage have identified multi-tiered and overlapping cellular systems that have evolved as a means of stabilizing the genome. One of these pathways supports DNA replication events by in a sense adopting the mantra that one must “make the best of a bad situation” and tolerating covalent modification to DNA through less accurate copying of the damaged region. Part of this so-called DNA damage tolerance pathway involves the recruitment of specialized DNA polymerases to sites of stalled or collapsed replication forks. These enzymes have unique structural and functional attributes that often allow bypass of adducted template DNA and successful completion of genomic replication. What follows is a selective description of the salient structural features and bypass properties of specialized DNA polymerases with an emphasis on Y-family members.

  14. Oxidatively generated base damage to cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, Jean [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no.3 - CEA/UJF, Institut nano-sciences et Cryogenie, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France); Departement de Medecine Nucleaire et Radiobiologie, Faculte de medecine de des sciences de la sante, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada); Douki, Thierry; Ravanat, Jean-Luc [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no.3 - CEA/UJF, Institut nano-sciences et Cryogenie, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France)

    2010-07-01

    Search for the formation of oxidatively base damage in cellular DNA has been a matter of debate for more than 40 years due to the lack of accurate methods for the measurement of the lesions. HPLC associated with either tandem mass spectrometry (MS/MS) or electrochemical detector (ECD) together with optimized DNA extraction conditions constitutes a relevant analytical approach. This has allowed the accurate measurement of oxidatively generated single and clustered base damage in cellular DNA following exposure to acute oxidative stress conditions mediated by ionizing radiation. UVA light and one-electron oxidants. In this review the formation of 11 single base lesions that is accounted for by reactions of singlet oxygen, hydroxyl radical or high intensity UVC laser pulses with nucleobases is discussed on the basis of the mechanisms available from model studies. In addition several clustered lesions were found to be generated in cellular DNA as the result of one initial radical hit on either a vicinal base or the 2-deoxyribose. Information on nucleo-base modifications that are formed upon addition of reactive aldehydes arising from the breakdown of lipid hydroperoxides is also provided. (authors)

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

    OpenAIRE

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

    2012-01-01

    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 ...

  16. Targeting Ongoing DNA Damage in Multiple Myeloma: Effects of DNA Damage Response Inhibitors on Plasma Cell Survival

    Directory of Open Access Journals (Sweden)

    Ana Belén Herrero

    2017-05-01

    Full Text Available Human myeloma cell lines (HMCLs and a subset of myeloma patients with poor prognosis exhibit high levels of replication stress (RS, leading to DNA damage. In this study, we confirmed the presence of DNA double-strand breaks (DSBs in several HMCLs by measuring γH2AX and RAD51 foci and analyzed the effect of various inhibitors of the DNA damage response on MM cell survival. Inhibition of ataxia telangiectasia and Rad3-related protein (ATR, the main kinase mediating the response to RS, using the specific inhibitor VE-821 induced more cell death in HMCLs than in control lymphoblastoid cells and U266, an HMCL with a low level of DNA damage. The absence of ATR was partially compensated by ataxia telangiectasia-mutated protein (ATM, since chemical inhibition of both kinases using VE-821 and KU-55933 significantly increased the death of MM cells with DNA damage. We found that ATM and ATR are involved in DSB repair by homologous recombination (HR in MM. Inhibition of both kinases resulted in a stronger inhibition that may underlie cell death induction, since abolition of HR using two different inhibitors severely reduced survival of HMCLs that exhibit DNA damage. On the other hand, inhibition of the other route involved in DSB repair, non-homologous end joining (NHEJ, using the DNA-PK inhibitor NU7441, did not affect MM cell viability. Interestingly, we found that NHEJ inhibition did not increase cell death when HR was simultaneously inhibited with the RAD51 inhibitor B02, but it clearly increased the level of cell death when HR was inhibited with the MRE11 inhibitor mirin, which interferes with recombination before DNA resection takes place. Taken together, our results demonstrate for the first time that MM cells with ongoing DNA damage rely on an intact HR pathway, which thereby suggests therapeutic opportunities. We also show that inhibition of HR after the initial step of end resection might be more appropriate for inducing MM cell death, since it

  17. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  18. Mechanisms of dealing with DNA damage in terminally differentiated cells

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

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

    Science.gov (United States)

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

    2015-08-01

    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 2 weeks in vivo. We show that influenza induces DNA damage to 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 DNA double strand break (DSB) repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA damage 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.

  20. Noncoding RNAs in DNA Damage Response: Opportunities for Cancer Therapeutics.

    Science.gov (United States)

    Arjumand, Wani; Asiaf, Asia; Ahmad, Shiekh Tanveer

    2018-01-01

    DNA repair machinery preserves genomic integrity, which is frequently challenged through endogenous and exogenous toxic insults, and any sort of repair machinery malfunctioning ultimately manifests in the form of several types of terrible human diseases such as cancers (Hoeijmakers, Nature 411(6835): 366-374, 2001). Noncoding RNAs (ncRNAs) are crucial players of DNA repair machinery in a cell and play a vital role in maintaining genomic stability, which is essential for its survival and normal functioning thus preventing tumorigenesis. To preserve the integrity of the genome, cells initiate a specific cellular response, recognized as DNA damage response (DDR), which includes several distinct DNA repair pathways. These repair pathways permit normal cells to repair DNA damage or induce apoptosis and cell cycle arrest in case the damage is irreparable. Disruption of these pathways in cancer leads to an increase in genomic instability and mutagenesis. Recently, emerging evidence suggests that ncRNAs play a critical role in the regulation of DDR. There is an extensive crosstalk between ncRNAs and the canonical DDR signaling pathway. DDR-induced expression of ncRNAs can provide a regulatory mechanism to accurately control the expression of DNA damage responsive genes in a spatio-temporal manner. DNA damage alters expression of a variety of ncRNAs at multiple levels including transcriptional regulation, post-transcriptional regulation, and RNA degradation and vice versa, wherein ncRNAs can directly regulate cellular processes involved in DDR by altering expression of their targeting genes, with a particular emphasis on microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). Relationship between the defects in the DDR and deregulation of related ncRNAs in human cancers is one of the established, which is growing stronger with the advent of high-throughput sequencing techniques such as next-generation sequencing. Understanding of the mechanisms that explain the association

  1. Repair of endogenous and ionizing radiation-induced DNA damages: mechanisms and biological functions

    International Nuclear Information System (INIS)

    Boiteux, S.

    2002-01-01

    The cellular DNA is continuously exposed to endogenous and exogenous stress. Oxidative stress due to cellular metabolism is the major cause of endogenous DNA damage. On the other hand, ionizing radiation (IR) is an important exogenous stress. Both induce similar DNA damages: damaged bases, abasic sites and strand breakage. Most of these lesions are lethal and/or mutagenic. The survival of the cell is managed by efficient and accurate DNA repair mechanisms that remove lesions before their replication or transcription. DNA repair pathways involved in the removal of IR-induced lesions are briefly described. Base excision repair (BER) is mostly involved in the removal of base damage, abasic sites and single strand breaks. In contrast, DNA double strand breaks are mostly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). How DNA repair pathways prevent cancer process is also discussed. (author)

  2. Solar radiation and mitochondrial DNA damage

    International Nuclear Information System (INIS)

    Hill, H.Z.; Locitzer, J.; Nassrin, E.; Ogbonnaya, A.; Hubbard, K.

    2003-01-01

    The 16.6 kB human mitochondrial DNA contains two homologous 13 base pair direct repeats separated by about 5 kB. During asynchronous mitochondrial DNA replication, the distant repeat sequences are thought to anneal, resulting in the looping out of a portion of the non-template strand which is subsequently deleted as a result of interaction with reactive oxygen species (ROS). A normal daughter and a deleted daughter mitochondrion result from such insults. This deletion has been termed the common deletion as it is the most frequent of the known mitochondrial DNA deletions. The common deletion is present in high frequency in several mitochondrial disorders, accumulates with age in slow turnover tissues and is increased in sun-exposed skin. Berneburg, et al. (Photochem. Photobiol. 66: 271, 1997) induced the common deletion in normal human fibroblasts after repeated exposures to UVA. In this study, the common deletion has been shown to be induced by repeated non-lethal exposures to FS20 sunlamp irradiation. Increases in the common deletion were demonstrated using nested PCR which produced a 303 bp product that was compared to a 324 bp product that required the presence of the undeleted 5 kB region. The cells were exposed to 10 repeated doses ranging from 0.5 (UVB) - 0.24 (UVA) J/sq m to 14.4 (UVB) - 5.8 J/sq m (UVA) measured using a UVX digital radiometer and UVB and UVA detectors respectively. Comparison with the earlier study by Berneberg, et al. suggests that this type of simulated solar damage is considerably more effective in fewer exposures than UVA radiation alone. The common deletion provides a cytoplasmic end-point for ROS damage produced by low dose chronic irradiations and other low level toxic exposures and should prove useful in evaluating cytoplasmic damage produced by ionizing radiation as well

  3. Protection of DNA damage by radiation exposure

    International Nuclear Information System (INIS)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents

  4. Protection of DNA damage by radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents.

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

    Science.gov (United States)

    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. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  6. DNA Damage Response and Immune Defence: Links and Mechanisms

    Directory of Open Access Journals (Sweden)

    Björn Schumacher

    2016-08-01

    Full Text Available 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 signalling 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 signalling. We highlight evidence gained into (i which molecular and cellular pathways of DDR activate immune signalling, (ii how DNA damage drives chronic inflammation, and (iii how chronic inflammation causes DNA damage and pathology in humans.

  7. Influence of the presence of B chromosomes on DNA damage in Crepis capillaris.

    Directory of Open Access Journals (Sweden)

    Jolanta Kwasniewska

    Full Text Available The sensitivity of different plant species to mutagenic agents is related to the DNA content and organization of the chromatin, which have been described in ABCW and bodyguard hypotheses, respectively. Plant species that have B chromosomes are good models for the study of these hypotheses. This study presents an analysis of the correlation between the occurrence of B chromosomes and the DNA damage that is induced by the chemical mutagen, maleic hydrazide (MH, in Crepis capillaris plants using comet assay. The presence of B chromosomes has a detectable impact on the level of DNA damage. The level of DNA damage after MH treatment was correlated with the number of B chromosomes and it was observed that it increased significantly in plants with 3B chromosomes. We did not find evidence of the protective role from chemical mutagens of the constitutive heterochromatin for euchromatin in relation to DNA damage. The DNA damage involving the 25S rDNA sequences was analyzed using the comet-FISH technique. Fragmentation within or near the 25S rDNA involved the loci on the A and B chromosomes. The presence of B chromosomes in C. capillaris cells had an influence on the level of DNA damage that involves the 25S rDNA region.

  8. MicroRNAs, the DNA damage response and cancer

    International Nuclear Information System (INIS)

    Wouters, Maikel D.; Gent, Dik C. van; Hoeijmakers, Jan H.J.; Pothof, Joris

    2011-01-01

    Many carcinogenic agents such as ultra-violet light from the sun and various natural and man-made chemicals act by damaging the DNA. To deal with these potentially detrimental effects of DNA damage, cells induce a complex DNA damage response (DDR) that includes DNA repair, cell cycle checkpoints, damage tolerance systems and apoptosis. This DDR is a potent barrier against carcinogenesis and defects within this response are observed in many, if not all, human tumors. DDR defects fuel the evolution of precancerous cells to malignant tumors, but can also induce sensitivity to DNA damaging agents in cancer cells, which can be therapeutically exploited by the use of DNA damaging treatment modalities. Regulation of and coordination between sub-pathways within the DDR is important for maintaining genome stability. Although regulation of the DDR has been extensively studied at the transcriptional and post-translational level, less is known about post-transcriptional gene regulation by microRNAs, the topic of this review. More specifically, we highlight current knowledge about DNA damage responsive microRNAs and microRNAs that regulate DNA damage response genes. We end by discussing the role of DNA damage response microRNAs in cancer etiology and sensitivity to ionizing radiation and other DNA damaging therapeutic agents.

  9. Molecular mechanisms in radiation damage to DNA: Final report

    International Nuclear Information System (INIS)

    Osman, R.

    1996-01-01

    The objectives of this work were to elucidate the molecular mechanisms that were responsible for radiation-induced DNA damage. The studies were based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA

  10. DNA Damage-related Vascular Dysfunction: Pathways and Interventions

    NARCIS (Netherlands)

    P.K. Bautista-Niño (Paula)

    2017-01-01

    markdownabstractIn my thesis the role of DNA damage on vascular function was studied. DNA damage is one of the primary causes of aging, which is the strongest independent risk factor for chronic diseases such as cancer and cardiovascular diseases. Mice with defective DNA repair are excellent

  11. DNA damage-induced inflammation and nuclear architecture.

    Science.gov (United States)

    Stratigi, Kalliopi; Chatzidoukaki, Ourania; Garinis, George A

    2017-07-01

    Nuclear architecture and the chromatin state affect most-if not all- DNA-dependent transactions, including the ability of cells to sense DNA lesions and restore damaged DNA back to its native form. Recent evidence points to functional links between DNA damage sensors, DNA repair mechanisms and the innate immune responses. The latter raises the question of how such seemingly disparate processes operate within the intrinsically complex nuclear landscape and the chromatin environment. Here, we discuss how DNA damage-induced immune responses operate within chromatin and the distinct sub-nuclear compartments highlighting their relevance to chronic inflammation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    Science.gov (United States)

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  13. DNA Damage Repair System in Plants: A Worldwide Research Update.

    Science.gov (United States)

    Gimenez, Estela; Manzano-Agugliaro, Francisco

    2017-10-30

    Living organisms are usually exposed to various DNA damaging agents so the mechanisms to detect and repair diverse DNA lesions have developed in all organisms with the result of maintaining genome integrity. Defects in DNA repair machinery contribute to cancer, certain diseases, and aging. Therefore, conserving the genomic sequence in organisms is key for the perpetuation of life. The machinery of DNA damage repair (DDR) in prokaryotes and eukaryotes is similar. Plants also share mechanisms for DNA repair with animals, although they differ in other important details. Plants have, surprisingly, been less investigated than other living organisms in this context, despite the fact that numerous lethal mutations in animals are viable in plants. In this manuscript, a worldwide bibliometric analysis of DDR systems and DDR research in plants was made. A comparison between both subjects was accomplished. The bibliometric analyses prove that the first study about DDR systems in plants (1987) was published thirteen years later than that for other living organisms (1975). Despite the increase in the number of papers about DDR mechanisms in plants in recent decades, nowadays the number of articles published each year about DDR systems in plants only represents 10% of the total number of articles about DDR. The DDR research field was done by 74 countries while the number of countries involved in the DDR & Plant field is 44. This indicates the great influence that DDR research in the plant field currently has, worldwide. As expected, the percentage of studies published about DDR systems in plants has increased in the subject area of agricultural and biological sciences and has diminished in medicine with respect to DDR studies in other living organisms. In short, bibliometric results highlight the current interest in DDR research in plants among DDR studies and can open new perspectives in the research field of DNA damage repair.

  14. Melanogenesis: a photoprotective response to DNA damage?

    International Nuclear Information System (INIS)

    Agar, Nita; Young, Antony R.

    2005-01-01

    Exposure to ultra violet radiation (UVR) is associated with significant long-term deleterious effects such as skin cancer. A well-recognised short-term consequence of UVR is increased skin pigmentation. Pigmentation, whether constitutive or facultative, has widely been viewed as photoprotective, largely because darkly pigmented skin is at a lower risk of photocarcinogenesis than fair skin. Research is increasingly suggesting that the relationship between pigmentation and photoprotection may be far more complex than previously assumed. For example, photoprotection against erythema and DNA damage has been shown to be independent of level of induced pigmentation in human white skin types. Growing evidence now suggests that UVR induced DNA photodamage, and its repair is one of the signals that stimulates melanogenesis and studies suggest that repeated exposure in skin type IV results in faster DNA repair in comparison to skin type II. These findings suggest that tanning may be a measure of inducible DNA repair capacity, and it is this rather than pigment per se which results in the lower incidence skin cancer observed in darker skinned individuals. This evokes the notion that epidermal pigmentation may in fact be the mammalian equivalent of a bacterial SOS response. Skin colour is one of most conspicuous ways in which humans vary yet the function of melanin remains controversial. Greater understanding of the role of pigmentation in skin is vital if one is to be able to give accurate advice to the general public about both the population at risk of skin carcinogenesis and also public perceptions of a tan as being healthy

  15. Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

    Science.gov (United States)

    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

  16. Aging and oxidatively damaged nuclear DNA in animal organs

    DEFF Research Database (Denmark)

    Møller, Peter; Løhr, Mille; Folkmann, Janne K

    2010-01-01

    with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies......Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs...... reporting a baseline level of damaged DNA that was fewer than 5 lesions/10(6) dG showed that 21 of 29 studies reported age-associated accumulation of DNA damage. The standardized mean difference in oxidatively damaged DNA between the oldest and the youngest age groups was 1.49 (95% CI 1...

  17. Breaking the DNA damage response to improve cervical cancer treatment.

    Science.gov (United States)

    Wieringa, Hylke W; van der Zee, Ate G J; de Vries, Elisabeth G E; van Vugt, Marcel A T M

    2016-01-01

    Every year, cervical cancer affects ∼500,000 women worldwide, and ∼275,000 patients die of this disease. The addition of platin-based chemotherapy to primary radiotherapy has increased 5-year survival of advanced-stage cervical cancer patients, which is, however, still only 66%. One of the factors thought to contribute to treatment failure is the ability of tumor cells to repair chemoradiotherapy-induced DNA damage. Therefore, sensitization of tumor cells for chemoradiotherapy via inhibition of the DNA damage response (DDR) as a novel strategy to improve therapy effect, is currently studied pre-clinically as well as in the clinic. Almost invariably, cervical carcinogenesis involves infection with the human papillomavirus (HPV), which inactivates part of the DNA damage response. This HPV-mediated partial inactivation of the DDR presents therapeutic targeting of the residual DDR as an interesting approach to achieve chemoradio-sensitization for cervical cancer. How the DDR can be most efficiently targeted, however, remains unclear. The fact that cisplatin and radiotherapy activate multiple signaling axes within the DDR further complicates a rational choice of therapeutic targets within the DDR. In this review, we provide an overview of the current preclinical and clinical knowledge about targeting the DDR in cervical cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Lans Hannes

    2012-01-01

    Full Text Available Abstract 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.

  1. A review and appraisal of the DNA damage theory of ageing.

    Science.gov (United States)

    Freitas, Alex A; de Magalhães, João Pedro

    2011-01-01

    Given the central role of DNA in life, and how ageing can be seen as the gradual and irreversible breakdown of living systems, the idea that damage to the DNA is the crucial cause of ageing remains a powerful one. DNA damage and mutations of different types clearly accumulate with age in mammalian tissues. Human progeroid syndromes resulting in what appears to be accelerated ageing have been linked to defects in DNA repair or processing, suggesting that elevated levels of DNA damage can accelerate physiological decline and the development of age-related diseases not limited to cancer. Higher DNA damage may trigger cellular signalling pathways, such as apoptosis, that result in a faster depletion of stem cells, which in turn contributes to accelerated ageing. Genetic manipulations of DNA repair pathways in mice further strengthen this view and also indicate that disruption of specific pathways, such as nucleotide excision repair and non-homologous end joining, is more strongly associated with premature ageing phenotypes. Delaying ageing in mice by decreasing levels of DNA damage, however, has not been achieved yet, perhaps due to the complexity inherent to DNA repair and DNA damage response pathways. Another open question is whether DNA repair optimization is involved in the evolution of species longevity, and we suggest that the way cells from different organisms respond to DNA damage may be crucial in species differences in ageing. Taken together, the data suggest a major role of DNA damage in the modulation of longevity, possibly through effects on cell dysfunction and loss, although understanding how to modify DNA damage repair and response systems to delay ageing remains a crucial challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Single Molecule Scanning of DNA Radiation Oxidative Damage Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  3. Single Molecule Scanning of DNA Radiation Oxidative Damage, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Natural transformation of bacteria by fragmented, damaged and ancient DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    . The degrading DNA is fragmented and damaged, often to less than one hundred base pairs. Such DNA is only recognized as microbial nutrients and is not considered as direct contributors to bacterial evolutionary processes. The main study shows natural transformation by very short DNA (≥20bp). Further we also show...... it by damaged short DNA with abasic sites, crosslinks, and miscoding lesions, which are the most common damages in environmental DNA. This is emphasized by successful natural transformation by 43,000-year-old DNA. We find that the process is a simple variant of natural transformation. On top, we illustrate...... with fullgenome comparisons that the process has general relevance in extant bacteria. Our findings reveal that the large environmental reservoir of short and damaged DNA retains capacity for natural transformation, even after thousands of years. This describes for the first time a process by which cells can...

  7. Repair of UV-damaged incoming plasmid DNA in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Keszenman-Pereyra, David

    1990-01-01

    A whole-cell transformation assay was used for the repair of UV-damaged plasma DNA in highly-transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities. The experiments described demonstrate that three epistasis groups (Friedberg 1988) are involved in the repair of UV-incoming DNA and that the repair processes act less efficiently on incoming DNA than they do on chromosomal DNA. The implications of these findings for UV repair in Saccharomyces cerevisiae are discussed. (author)

  8. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.

    2013-01-01

    Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time...

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

    Science.gov (United States)

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

    2012-07-06

    DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Chromatin modifications and the DNA damage response to ionizing radiation

    International Nuclear Information System (INIS)

    Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

    2013-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

  11. Sperm DNA damage in relation to lipid peroxidation following ...

    African Journals Online (AJOL)

    This study investigated the relationships between lipid peroxidation (LPO) and sperm DNA damage following freezing-thawing of boar semen in different extenders. The comet assay was used to measure the extent of sperm DNA damage in a cryoprotectant-free extender or in cryoprotectant-based extenders after single ...

  12. DNA damage protection and 5-lipoxygenase inhibiting activity of ...

    African Journals Online (AJOL)

    DNA damage caused by free radical is associated with mutation-based health impairment. The protective effect on DNA damage mediated by hydroxyl radical and peroxynitrite radical, and the inhibiting activity on 5-lipoxygenase of areca inflorescence extracts were studied in vitro. The results show that the boiling water ...

  13. Assessment of DNA damage by panmasala, gutkha chewing and ...

    African Journals Online (AJOL)

    In the present study the comet assay was performed in buccal epithelial cells to evaluate DNA damage among pan masala or gutkha chewers and smokers. The assay is a rapid, suitable and sensitive method for detecting various forms of DNA damage at individual cell level. The study comprises 300 individuals of which 50 ...

  14. Assessment of DNA damage by panmasala, gutkha chewing and ...

    African Journals Online (AJOL)

    Smita Jyoti

    2013-09-04

    Sep 4, 2013 ... Abstract In the present study the comet assay was performed in buccal epithelial cells to evaluate. DNA damage among pan masala or gutkha chewers and smokers. The assay is a rapid, suitable and sensitive method for detecting various forms of DNA damage at individual cell level. The study comprises ...

  15. DNA damage response and Autophagy: a meaningful partnership

    Directory of Open Access Journals (Sweden)

    ARISTIDES G ELIOPOULOS

    2016-11-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

  17. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could...

  18. DNA mismatch repair and the DNA damage response to ionizing radiation: making sense of apparently conflicting data.

    Science.gov (United States)

    Martin, Lynn M; Marples, Brian; Coffey, Mary; Lawler, Mark; Lynch, Thomas H; Hollywood, Donal; Marignol, Laure

    2010-11-01

    The DNA mismatch repair (MMR) pathway detects and repairs DNA replication errors. While DNA MMR-proficiency is known to play a key role in the sensitivity to a number of DNA damaging agents, its role in the cytotoxicity of ionizing radiation (IR) is less well characterized. Available literature to date is conflicting regarding the influence of MMR status on radiosensitivity, and this has arisen as a subject of controversy in the field. The aim of this paper is to provide the first comprehensive overview of the experimental data linking MMR proteins and the DNA damage response to IR. A PubMed search was conducted using the key words "DNA mismatch repair" and "ionizing radiation". Relevant articles and their references were reviewed for their association between DNA MMR and IR. Recent data suggest that radiation dose and the type of DNA damage induced may dictate the involvement of the MMR system in the cellular response to IR. In particular, the literature supports a role for the MMR system in DNA damage recognition, cell cycle arrest, DNA repair and apoptosis. In this review we discuss our current understanding of the impact of MMR status on the cellular response to radiation in mammalian cells gained from past and present studies and attempt to provide an explanation for how MMR may determine the response to radiation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Response to DNA damage: why do we need to focus on protein phosphatases?

    Directory of Open Access Journals (Sweden)

    Midori eShimada

    2013-01-01

    Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

  20. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

    Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2017-10-01

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

  3. Primary DNA Damage in Dry Cleaners with Perchlorethylene Exposure

    Directory of Open Access Journals (Sweden)

    Mohammad Azimi

    2017-10-01

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

  4. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp.

    NARCIS (Netherlands)

    Buma, A.G.J.; van Hannen, E.J; Veldhuis, M.J W; Gieskes, W.W C

    The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

  5. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

    NARCIS (Netherlands)

    Buma, A.G.J.; Van Hannen, E.J.; Veldhuis, M.; Gieskes, W.W.C.

    1996-01-01

    The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

  6. Cancer risk and oxidative DNA damage in man

    DEFF Research Database (Denmark)

    Loft, Steffen; Poulsen, H E

    1996-01-01

    with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion...... of Brussels sprouts reduced the oxidative DNA damage rate, estimated by the urinary excretion of 8-oxodG, and the intake of vitamin C was a determinant for the level of 8-oxodG in sperm DNA. A low-fat diet reduced another marker of oxidative DNA damage in leukocytes. In patients with diseases associated...... of biobank material using a nested case control design. In addition, oxidative damage may be important for the aging process, particularly with respect to mitochondrial DNA and the pathogenesis of inflammatory diseases....

  7. Cancer risk and oxidative DNA damage in man

    DEFF Research Database (Denmark)

    Loft, Steffen; Poulsen, H E

    1996-01-01

    of ROS. These include oxidative damage to DNA, which experimental studies in animals and in vitro have suggested are an important factor in carcinogenesis. Despite extensive repair oxidatively modified DNA is abundant in human tissues, in particular in tumors, i.e., in terms of 1-200 modified nucleosides...... per 10(5) intact nucleosides. The damaged nucleosides accumulate with age in both nuclear and mitochondrial DNA. The products of repair of these lesions are excreted into the urine in amounts corresponding to a damage rate of up to 10(4) modifications in each cell every day. The most abundant...... and their biological significance less apparent. The biomarkers for study of oxidative DNA damage in humans include urinary excretion of oxidized nucleosides and bases as repair products and modifications in DNA isolated from target tissue or surrogate cells, such as lymphocytes. These biomarkers reflect the rate...

  8. Bacterial natural transformation by highly fragmented and damaged DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic Antoine Alexandre

    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 f...... 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.......DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often 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...

  9. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

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

  10. Role of the Checkpoint Clamp in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2013-01-01

    Full Text Available DNA damage occurs during DNA replication, spontaneous chemical reactions, and assaults by external or metabolism-derived agents. Therefore, all living cells must constantly contend with DNA damage. Cells protect themselves from these genotoxic stresses by activating the DNA damage checkpoint and DNA repair pathways. Coordination of these pathways requires tight regulation in order to prevent genomic instability. The checkpoint clamp complex consists of Rad9, Rad1 and Hus1 proteins, and is often called the 9-1-1 complex. This PCNA (proliferating cell nuclear antigen-like donut-shaped protein complex is a checkpoint sensor protein that is recruited to DNA damage sites during the early stage of the response, and is required for checkpoint activation. As PCNA is required for multiple pathways of DNA metabolism, the checkpoint clamp has also been implicated in direct roles in DNA repair, as well as in coordination of the pathways. Here we discuss roles of the checkpoint clamp in DNA damage response (DDR.

  11. Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

    Science.gov (United States)

    Nair, Nidhi; Shoaib, Muhammad

    2017-01-01

    Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage. PMID:28698521

  12. DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

    Science.gov (United States)

    Dutertre, Martin; Vagner, Stéphan

    2017-10-27

    Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

    International Nuclear Information System (INIS)

    Cooke, Marcus S.; Evans, Mark D.; Dove, Rosamund; Rozalski, Rafal; Gackowski, Daniel; Siomek, Agnieszka; Lunec, Joseph; Olinski, Ryszard

    2005-01-01

    The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15 N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair

  14. Sensing DNA damage by PARP-like fingers

    OpenAIRE

    Petrucco, Stefania

    2003-01-01

    PARP-like zinc fingers are protein modules, initially described as nick-sensors of poly(ADP-ribosyl)-polymerases (PARPs), which are found at the N-terminus of different DNA repair enzymes. I chose to study the role of PARP-like fingers in AtZDP, a 3′ DNA phosphoesterase, which is the only known enzyme provided with three such finger domains. Here I show that PARP-like fingers can maintain AtZDP onto damaged DNA sites without interfering with its DNA end repair functions. Damage recognition by...

  15. DNA Damage and Base Excision Repair in Mitochondria and Their Role in Aging

    Directory of Open Access Journals (Sweden)

    Ricardo Gredilla

    2011-01-01

    Full Text Available During the last decades, our knowledge about the processes involved in the aging process has exponentially increased. However, further investigation will be still required to globally understand the complexity of aging. Aging is a multifactorial phenomenon characterized by increased susceptibility to cellular loss and functional decline, where mitochondrial DNA mutations and mitochondrial DNA damage response are thought to play important roles. Due to the proximity of mitochondrial DNA to the main sites of mitochondrial-free radical generation, oxidative stress is a major source of mitochondrial DNA mutations. Mitochondrial DNA repair mechanisms, in particular the base excision repair pathway, constitute an important mechanism for maintenance of mitochondrial DNA integrity. The results reviewed here support that mitochondrial DNA damage plays an important role in aging.

  16. Mechanisms for radiation damage in DNA. Progress report, June 1, 1994--May 31, 1995

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1994-11-01

    In this project we have proposed several mechanisms for radiation damage to DNA and its constituents, and have detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. The results from these various techniques have resulted in an understanding of consequences of radiation damage to DNA from the early ionization event to the production of non-radical lesions (discussed in detail in Comprehensive Report). In this year's work we have found the hydroxyl radical in DNA's hydration layer. This is an important result which impacts the hole transfer hypothesis and the understanding of the direct vs. indirect effect in DNA. Further we have found the first ESR evidence for sugar radicals as a result of direct radiation damage to DNA nucleotides in an aqueous environment. This is significant as it impacts the biological endpoint of radiation damage to DNA and suggests future work in DNA. Work with DNA-polypeptides show clear evidence for electron transfer to DNA from the polypeptide which we believe is a radioprotective mechanism. Our work with ab initio molecular orbital theory has gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics involved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this year's work to new, more accurate values for the electron affinities of the DNA bases, understanding of the relative stability of all possible sugar radicals formed by hydrogen abstraction on the deoxyribose group, hydration effects on, thiol radioprotectors, and an ongoing study of radical intermediates formed from initial DNA ion radicals. During this fiscal year five articles have been published, three are in press, two are submitted and several more are in preparation

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

    Science.gov (United States)

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

    2017-05-01

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

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

    National Research Council Canada - National Science Library

    Thompson, Henry

    2000-01-01

    The purpose of this project is to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

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

    National Research Council Canada - National Science Library

    Thompson, Henry

    2003-01-01

    The purpose of this project was to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

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

    National Research Council Canada - National Science Library

    Thompson, Henry

    2002-01-01

    The purpose of this project is to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

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

    National Research Council Canada - National Science Library

    Thompson, Henry

    2001-01-01

    The purpose of this project is to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

  2. DNA damage caused by UV- and near UV-irradiation

    International Nuclear Information System (INIS)

    Ohnishi, Takeo

    1986-01-01

    Much work with mutants deficient in DNA repair has been performed concerning UV-induced DNA damage under the condition where there is no artificial stimulation. In an attempt to infer the effects of solar wavelengths, the outcome of the work is discussed in terms of cellular radiation sensitivity, unscheduled DNA synthesis, and mutation induction, leading to the conclusion that some DNA damage occurs even by irradiation of the shorter wavelength light (270 - 315 nm) and is repaired by excision repair. It has been thought to date that pyrimidine dimer (PD) plays the most important role in UV-induced DNA damage, followed by (6 - 4) photoproducts. As for DNA damage induced by near UV irradiation, the yield of DNA single-strand breaks and of DNA-protein crosslinking, other than PD, is considered. The DNA-protein crosslinking has proved to be induced by irradiation at any wavelength of UV ranging from 260 to 425 nm. Near UV irradiation causes the inhibition of cell proliferation to take place. (Namekawa, K.)

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

    Science.gov (United States)

    Hu, Jinchuan; Adar, Sheera

    2017-01-01

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

  4. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  5. Primary DNA Damage in Dry Cleaners with Perchlorethylene Exposure

    OpenAIRE

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

    2017-01-01

    Background: Perchloroethylene is a halogenated solvent widely used in dry cleaning. International agency of research on cancer classified this chemical as a probable human carcinogen. Objective: To evaluate the extent of primary DNA damage in dry cleaner workers who were exposed to perchloroethylene as compared to non-exposed subjects. The effect of exposure modifying factors such as use of personal protective equipment, perceived risk, and reported safe behaviors on observed DNA damage w...

  6. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  7. Coupling mechanisms between nucleosome assembly and the cellular response to DNA damage

    International Nuclear Information System (INIS)

    Lautrette, Aurelie

    2006-01-01

    Cells are continuously exposed to genotoxic stresses that induce a variety of DNA lesions. To protect their genome, cells have specific pathways that orchestrate the detection, signaling and repair of DNA damages. This work is dedicated to the characterization of such pathways that couple the DNA damage response to the assembly of chromatin, a complex that protects and regulates DNA accessibility. We have focused our study on two multifunctional proteins: Rad53, a central checkpoint kinase in the cellular response to DNA damage and Asf1, a histone chaperone involved in chromatin assembly. We have characterized in vitro the binding mode of Asf1 with Rad53 and Asfl with histones. This study is associated with the functional analysis of the role of these interactions in vivo in yeast cells. (author) [fr

  8. Non coding RNA: sequence-specific guide for chromatin modification and DNA damage signaling

    Directory of Open Access Journals (Sweden)

    Sofia eFrancia

    2015-11-01

    Full Text Available Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports suggest that ncRNAs are involved in DDR signaling and homology-mediated DNA repair. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair.

  9. Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

    Science.gov (United States)

    Garcia, Juliana B F; Rocha, João P Vieira da; Costa-Silva, Héllida M; Alves, Ceres L; Machado, Carlos R; Cruz, Angela K

    2016-05-01

    Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Radiation damage to DNA: the effect of LET

    Energy Technology Data Exchange (ETDEWEB)

    Ward, J.F.; Milligan, J.R. [California Univ., San Diego, La Jolla, CA (United States). School of Medicine

    1997-03-01

    Mechanisms whereby ionizing radiation induced damage are introduced into cellular DNA are discussed. The types of lesions induced are summarized and the rationale is presented which supports the statement that radiation induced singly damaged sites are biologically unimportant. The conclusion that multiply damaged sites are critical is discussed and the mechanisms whereby such lesions are formed are presented. Structures of multiply damaged sites are summarized and problems which they present to cellular repair systems are discussed. Lastly the effects of linear energy transfer on the complexity of multiply damaged sites are surveyed and the consequences of this increased complexity are considered in terms of cell survival and mutation. (author)

  11. DNA damage response in a radiation resistant bacterium Deinococcus radiodurans: a paradigm shift

    International Nuclear Information System (INIS)

    Misra, H.S.

    2015-01-01

    Deinococcusradiodurans is best known for its extraordinary resistance to gamma radiation with its D 10 12kGy, and several other DNA damaging agents including desiccation to less than 5% humidity and chemical xenotoxicants. An efficient DNA double strand break (DSB) repair and its ability to protect biomolecules from oxidative damage are a few mechanisms attributed to these phenotypes in this bacterium. Although it regulates its proteome and transcriptome in response to DNA damage for its growth and survival, it lacks LexA mediated classical SOS response mechanism. Since LexA mediated damages response mechanism is highly and perhaps only, characterized DNA damage response processes in prokaryotes, this bacterium keeps us guessing how it responds to extreme doses of DNA damage. Interestingly, this bacterium encodes a large number of eukaryotic type serine threonine/tyrosine protein kinases (eST/YPK), phosphatases and response regulators and roles of eST/YPKs in cellular response to DNA damage and cell cycle regulations are well established in eukaryotes. Here, we characterized an antioxidant and DNA damage inducible eST/YPK (RqkA) and established its role in extraordinary radioresistance and DSB repair in this bacterium. We identified native phosphoprotein substrates for this kinase and demonstrated the involvement of some of these proteins phosphorylation in the regulation of DSB repair and growth under radiation stress. Findings suggesting the possible existence of eST/YPK mediated DNA damage response mechanism as an alternate to classical SOS response in this prokaryote would be discussed. (author)

  12. The complexity of DNA damage: relevance to biological consequences

    International Nuclear Information System (INIS)

    Ward, J.F.

    1994-01-01

    Ionizing radiation causes both singly and multiply damaged sites in DNA when the range of radical migration is limited by the presence of hydroxyl radical scavengers (e.g. within cells). Multiply damaged sites are considered to be more biologically relevant because of the challenges they present to cellular repair mechanisms. These sites occur in the form of DNA double-strand breaks (dsb) but also as other multiple damages that can be converted to dsb during attempted repair. The presence of a dsb can lead to loss of base sequence information and/or can permit the two ends of a break to separate and rejoin with the wrong partner. (Multiply damaged sites may also be the biologically relevant type of damage caused by other agents, such as UVA, B and/or C light, and some antitumour antibiotics). The quantitative data available from radiation studies of DNA are shown to support the proposed mechanisms for the production of complex damage in cellular DNA, i.e. via scavengable and non-scavengable mechanisms. The yields of complex damages can in turn be used to support the conclusion that cellular mutations are a consequence of the presence of these damages within a gene. (Author)

  13. Insight into the ERVK integrase – propensity for DNA damage

    Directory of Open Access Journals (Sweden)

    Samantha Bray

    2016-12-01

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

  14. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2005-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  15. Profiling DNA damage response following mitotic perturbations

    DEFF Research Database (Denmark)

    Pedersen, Ronni Sølvhøi; Karemore, Gopal; Gudjonsson, Thorkell

    2016-01-01

    Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show t...

  16. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2004-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  17. Choreography of the DNA damage response

    DEFF Research Database (Denmark)

    Lisby, Michael; Barlow, Jacqueline H; Burgess, Rebecca C

    2004-01-01

    recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting......DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication...... stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous...

  18. Vitamin C for DNA damage prevention

    Czech Academy of Sciences Publication Activity Database

    Šrám, Radim; Binková, B.; Rössner ml., Pavel

    2012-01-01

    Roč. 733, 1-2 (2012), s. 39-49 ISSN 0027-5107 R&D Projects: GA MŠk 2B08005; GA MŽP(CZ) SP/1B3/50/07 Institutional research plan: CEZ:AV0Z50390703 Keywords : Chromosomal aberrations * DNA adducts * DNA repair Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.902, year: 2012

  19. Role of oxidative DNA damage in genome instability and cancer

    International Nuclear Information System (INIS)

    Bignami, M.; Kunkel, T.

    2009-01-01

    Inactivation of mismatch repair (MMR) is associated with a dramatic genomic instability that is observed experimentally as a mutator phenotype and micro satellite instability (MSI). It has been implicit that the massive genetic instability in MMR defective cells simply reflects the accumulation of spontaneous DNA polymerase errors during DNA replication. We recently identified oxidation damage, a common threat to DNA integrity to which purines are very susceptible, as an important cofactor in this genetic instability

  20. Visualization of DNA clustered damage induced by heavy ion exposure

    International Nuclear Information System (INIS)

    Tomita, M.; Yatagai, F.

    2003-01-01

    Full text: DNA double-strand breaks (DSBs) are the most lethal damage induced by ionizing radiations. Accelerated heavy-ions have been shown to induce DNA clustered damage, which is two or more DNA lesions induced within a few helical turns. Higher biological effectiveness of heavy-ions could be provided predominantly by induction of complex DNA clustered damage, which leads to non-repairable DSBs. DNA-dependent protein kinase (DNA-PK) is composed of catalytic subunit (DNA-PKcs) and DNA-binding heterodimer (Ku70 and Ku86). DNA-PK acts as a sensor of DSB during non-homologous end-joining (NHEJ), since DNA-PK is activated to bind to the ends of double-stranded DNA. On the other hand, NBS1 and histone H2AX are essential for DSB repair by homologous recombination (HR) in higher vertebrate cells. Here we report that phosphorylated H2AX at Ser139 (named γ-H2AX) and NBS1 form large undissolvable foci after exposure to accelerated Fe ions, while DNA-PKcs does not recognize DNA clustered damage. NBS1 and γ-H2AX colocalized with forming discrete foci after exposure to X-rays. At 0.5 h after Fe ion irradiation, NBS1 and γ-H2AX also formed discrete foci. However, at 3-8 h after Fe ion irradiation, highly localized large foci turned up, while small discrete foci disappeared. Large NBS1 and γ-H2AX foci were remained even 16 h after irradiation. DNA-PKcs recognized Ku-binding DSB and formed foci shortly after exposure to X-rays. DNA-PKcs foci were observed 0.5 h after 5 Gy of Fe ion irradiation and were almost completely disappeared up to 8 h. These results suggest that NBS1 and γ-H2AX can be utilized as molecular marker of DNA clustered damage, while DNA-PK selectively recognizes repairable DSBs by NHEJ

  1. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

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

  2. Systemic DNA damage accumulation under in vivo tumor growth can be inhibited by the antioxidant Tempol.

    Science.gov (United States)

    Georgakilas, Alexandros G; Redon, Christophe E; Ferguson, Nicholas F; Kryston, Thomas B; Parekh, Palak; Dickey, Jennifer S; Nakamura, Asako J; Mitchell, James B; Bonner, William M; Martin, Olga A

    2014-10-28

    Recently we found that mice bearing subcutaneous non-metastatic tumors exhibited elevated levels of two types of complex DNA damage, i.e., double-strand breaks and oxidatively-induced clustered DNA lesions in various tissues throughout the body, both adjacent to and distant from the tumor site. This DNA damage was dependent on CCL2, a cytokine involved in the recruitment and activation of macrophages, suggesting that this systemic DNA damage was mediated via tumor-induced chronic inflammatory responses involving cytokines, activation of macrophages, and consequent free radical production. If free radicals are involved, then a diet containing an antioxidant may decrease the distant DNA damage. Here we repeated our standard protocol in cohorts of two syngeneic tumor-bearing C57BL/6NCr mice that were on a Tempol-supplemented diet. We show that double-strand break and oxidatively-induced clustered DNA lesion levels were considerably decreased, about two- to three fold, in the majority of tissues studied from the tumor-bearing mice fed the antioxidant Tempol compared to the control tumor-bearing mice. Similar results were also observed in nude mice suggesting that the Tempol effects are independent of functioning adaptive immunity. This is the first in vivo study demonstrating the effect of a dietary antioxidant on abscopal DNA damage in tissues distant from a localized source of genotoxic stress. These findings may be important for understanding the mechanisms of genomic instability and carcinogenesis caused by chronic stress-induced systemic DNA damage and for developing preventative strategies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-07-01

    This study aims to assess the effects of low-dose benzene on DNA damage and O 6 -methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers. We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay. The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage. MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.

  4. DNA damage and repair in age-related macular degeneration

    International Nuclear Information System (INIS)

    Szaflik, Jacek P.; Janik-Papis, Katarzyna; Synowiec, Ewelina; Ksiazek, Dominika; Zaras, Magdalena; Wozniak, Katarzyna; Szaflik, Jerzy; Blasiak, Janusz

    2009-01-01

    Age-related macular degeneration (AMD) is a retinal degenerative disease that is the main cause of vision loss in individuals over the age of 55 in the Western world. Clinically relevant AMD results from damage to the retinal pigment epithelial (RPE) cells thought to be mainly caused by oxidative stress. The stress also affects the DNA of RPE cells, which promotes genome instability in these cells. These effects may coincide with the decrease in the efficacy of DNA repair with age. Therefore individuals with DNA repair impaired more than average for a given age may be more susceptible to AMD if oxidative stress affects their RPE cells. This may be helpful in AMD risk assessment. In the present work we determined the level of basal (measured in the alkaline comet assay) endogenous and endogenous oxidative DNA damage, the susceptibility to exogenous mutagens and the efficacy of DNA repair in lymphocytes of 100 AMD patients and 110 age-matched individuals without visual disturbances. The cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage without differences between patients of dry and wet forms of the disease. DNA double-strand breaks did not contribute to the observed DNA damage as checked by the neutral comet assay and pulsed field gel electrophoresis. The extent of oxidative modification to DNA bases was grater in AMD patients than in the controls, as probed by DNA repair enzymes NTH1 and Fpg. Lymphocytes from AMD patients displayed a higher sensitivity to hydrogen peroxide and UV radiation and repaired lesions induced by these factors less effectively than the cells from the control individuals. We postulate that the impaired efficacy of DNA repair may combine with enhanced sensitivity of RPE cells to blue and UV lights, contributing to the pathogenesis of AMD.

  5. DNA damage and repair in age-related macular degeneration

    Energy Technology Data Exchange (ETDEWEB)

    Szaflik, Jacek P. [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Janik-Papis, Katarzyna; Synowiec, Ewelina; Ksiazek, Dominika [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Zaras, Magdalena [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Wozniak, Katarzyna [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Szaflik, Jerzy [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Blasiak, Janusz, E-mail: januszb@biol.uni.lodz.pl [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland)

    2009-10-02

    Age-related macular degeneration (AMD) is a retinal degenerative disease that is the main cause of vision loss in individuals over the age of 55 in the Western world. Clinically relevant AMD results from damage to the retinal pigment epithelial (RPE) cells thought to be mainly caused by oxidative stress. The stress also affects the DNA of RPE cells, which promotes genome instability in these cells. These effects may coincide with the decrease in the efficacy of DNA repair with age. Therefore individuals with DNA repair impaired more than average for a given age may be more susceptible to AMD if oxidative stress affects their RPE cells. This may be helpful in AMD risk assessment. In the present work we determined the level of basal (measured in the alkaline comet assay) endogenous and endogenous oxidative DNA damage, the susceptibility to exogenous mutagens and the efficacy of DNA repair in lymphocytes of 100 AMD patients and 110 age-matched individuals without visual disturbances. The cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage without differences between patients of dry and wet forms of the disease. DNA double-strand breaks did not contribute to the observed DNA damage as checked by the neutral comet assay and pulsed field gel electrophoresis. The extent of oxidative modification to DNA bases was grater in AMD patients than in the controls, as probed by DNA repair enzymes NTH1 and Fpg. Lymphocytes from AMD patients displayed a higher sensitivity to hydrogen peroxide and UV radiation and repaired lesions induced by these factors less effectively than the cells from the control individuals. We postulate that the impaired efficacy of DNA repair may combine with enhanced sensitivity of RPE cells to blue and UV lights, contributing to the pathogenesis of AMD.

  6. Study on DNA damages induced by UV radiation

    International Nuclear Information System (INIS)

    Doan Hong Van; Dinh Ba Tuan; Tran Tuan Anh; Nguyen Thuy Ngan; Ta Bich Thuan; Vo Thi Thuong Lan; Tran Minh Quynh; Nguyen Thi Thom

    2015-01-01

    DNA damages in Escherichia coli (E. coli) exposed to UV radiation have been investigated. After 30 min of exposure to UV radiation of 5 mJ/cm 2 , the growth of E. coli in LB broth medium was about only 10% in compared with non-irradiated one. This results suggested that the UV radiation caused the damages for E. coli genome resulted in reduction in its growth and survival, and those lesions can be somewhat recovered. For both solutions of plasmid DNAs and E. coli cells containing plasmid DNA, this dose also caused the breakage on single and double strands of DNA, shifted the morphology of DNA plasmid from supercoiled to circular and linear forms. The formation of pyrimidine dimers upon UV radiation significantly reduced when the DNA was irradiated in the presence of Ganoderma lucidum extract. Thus, studies on UV-induced DNA damage at molecular level are very essential to determine the UV radiation doses corresponding to the DNA damages, especially for creation and selection of useful radiation-induced mutants, as well as elucidation the protective effects of the specific compounds against UV light. (author)

  7. Histone modifications in response to DNA damage

    International Nuclear Information System (INIS)

    Altaf, Mohammed; Saksouk, Nehme; Cote, Jacques

    2007-01-01

    The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity

  8. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    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...... 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...

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

    Science.gov (United States)

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

    2016-08-01

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

  10. Radioadaptive response. Efficient repair of radiation-induced DNA damage in adapted cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji; Aritomi, Hisako; Morisita, Jun

    1996-01-01

    To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage

  11. DNA Repair and the Accumulation of Oxidatively Damaged DNA Are Affected by Fruit Intake in Mice

    DEFF Research Database (Denmark)

    Croteau, Deborah L; de Souza-Pinto, Nadja C; Harboe, Charlotte

    2010-01-01

    were fed for 14 weeks a control diet or a diet with 8% peach or nectarine extract. The activities of DNA repair enzymes, the level of DNA damage, and gene expression changes were measured. Our study showed that repair of various oxidative DNA lesions was more efficient in liver extracts derived from......Aging is associated with elevated oxidative stress and DNA damage. To achieve healthy aging, we must begin to understand how diet affects cellular processes. We postulated that fruit-enriched diets might initiate a program of enhanced DNA repair and thereby improve genome integrity. C57Bl/6 J mice......-fed mice. Taken together, these results suggest that an increased intake of fruits might modulate the efficiency of DNA repair, resulting in altered levels of DNA damage....

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

    Science.gov (United States)

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

    2013-12-03

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often 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.

  13. Hypochlorite-induced damage to DNA, RNA, and polynucleotides

    DEFF Research Database (Denmark)

    Hawkins, Clare Louise; Davies, Michael Jonathan

    2002-01-01

    HOCl damage to DNA, RNA, and polynucleotides. Reaction of HOCl with these materials is shown to yield multiple semistable chloramines (RNHCl/RR'NCl), which are the major initial products, and account for 50-95% of the added HOCl. These chloramines decay by thermal and metal-ion catalyzed processes...... favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA...... rationalize the preferential formation of chlorinated 2'-deoxycytidine and 2'-deoxyadenosine in DNA and suggest that DNA damage induced by HOCl, and preformed chloramines, occurs at sequence-specific sites....

  14. Viral DNA replication-dependent DNA damage response activation during BK polyomavirus infection.

    Science.gov (United States)

    Verhalen, Brandy; Justice, Joshua L; Imperiale, Michael J; Jiang, Mengxi

    2015-05-01

    BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral

  15. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

    2013-01-01

    DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed...... knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....

  16. DNA Damage Response in Hematopoietic Stem Cell Ageing.

    Science.gov (United States)

    Li, Tangliang; Zhou, Zhong-Wei; Ju, Zhenyu; Wang, Zhao-Qi

    2016-06-01

    Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity. Hematopoietic stem cells (HSCs) are the most primitive cell type in the hematopoietic system. They divide asymmetrically and give rise to daughter cells with HSC identity (self-renewal) and progenitor progenies (differentiation), which further proliferate and differentiate into full hematopoietic lineages. Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation. Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process. The DNA damage response (DDR) in the cells involves an orchestrated signaling pathway, consisting of cell cycle regulation, cell death and senescence, transcriptional regulation, as well as chromatin remodeling. Recent studies employing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically regulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system. In this review, we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  17. Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

    International Nuclear Information System (INIS)

    Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

    1988-01-01

    DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

  18. Evaluation of DNA damage using microwave dielectric absorption spectroscopy

    International Nuclear Information System (INIS)

    Hirayama, Makoto; Matuo, Youichrou; Izumi, Yoshinobu; Sunagawa, Takeyoshi

    2016-01-01

    Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pre-treatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy

  19. Evaluation of DNA damage using microwave dielectric absorption spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hirayama, Makoto; Matuo, Youichrou; Izumi, Yoshinobu [Research Institute of Nuclear Engineering, University of Fukui, Fukui (Japan); Sunagawa, Takeyoshi [Fukui University of Technology, Fukui (Japan)

    2016-12-15

    Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pre-treatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy.

  20. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

    Energy Technology Data Exchange (ETDEWEB)

    Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

    2004-11-22

    Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

  1. Topics in free radical-mediated DNA damage: purines and damage amplification - superoxic reactions - bleomycin, the incomplete radiomimetic

    International Nuclear Information System (INIS)

    Sonntag, C. von

    1994-01-01

    Only a small percentage of the DNA damage set by ionizing radiation in the living cell manifests itself as lethal. It is now increasingly accepted that clustered lesions may constitute the kind of damage that the repair enzymes cannot adequately deal with. The question is raised as to whether damage amplification reactions (radical transfer reactions) may contribute to these clustered lesions, and examples of such damage amplification reactions are given. In one example a purine is involved. With 2'-deoxy adenosine and 2'-deoxy guanosine it is shown that these purine nucleosides undergo unexpected radical reactions. Evidence for the radical transfer from the purine to the sugar moiety is provided by the formation of the 5'-aldehydes. These products have been assayed with 2-thiobarbituric acid (TBA), a reagent commonly applied to the detection of malonaldehyde. TBA-reactive material has also been assayed in γ-irradiated DNA, about one-third of this is free malonaldehyde, while the major part of the TBA-reactive material remains bound to the DNA. In contrast, bleomycin-treated DNA yields practically no free malonaldehyde, and the major TBA-reactive products are identified as the thymine and adenine base propenals. (Author)

  2. Radiation damage to DNA-protein complexes

    Czech Academy of Sciences Publication Activity Database

    Spotheim-Maurizot, M.; Davídková, Marie

    2011-01-01

    Roč. 261, zima (2011), s. 1-10 ISSN 1742-6588. [COST Chemistry CM0603-MELUSYN Joint Meeting Damages Induced in Biomolecules by Low and High Energy Radiations. Paříž, 09.03.2010-12.03.2010] R&D Projects: GA AV ČR IAA1048103; GA AV ČR KJB4048401; GA MŠk 1P05OC085; GA MŠk OC09012; GA AV ČR IAB1048901 Institutional research plan: CEZ:AV0Z10480505 Keywords : radiolysis * molecular-dynamics simulation * hydroxyl radical attack * induced strand breakage Subject RIV: BO - Biophysics

  3. Radiation damage to DNA in DNA-protein complexes

    Czech Academy of Sciences Publication Activity Database

    Spotheim Maurizot, M.; Davídková, Marie

    2011-01-01

    Roč. 711, 1-2 (2011), s. 41-48 ISSN 0027-5107 Institutional research plan: CEZ:AV0Z10480505 Keywords : DNA-protein complex * ionizing radiation * molecular structure Subject RIV: BO - Biophysics Impact factor: 2.850, year: 2011

  4. Histone peptide AKRHRK enhances H2O2-induced DNA damage and alters its site specificity

    International Nuclear Information System (INIS)

    Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke

    2005-01-01

    Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition

  5. Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

    Science.gov (United States)

    Korycka-Machala, Malgorzata; Brzostek, Anna; Rozalska, Sylwia; Rumijowska-Galewicz, Anna; Dziedzic, Renata; Bowater, Richard; Dziadek, Jaroslaw

    2006-05-01

    Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.

  6. An extended sequence specificity for UV-induced DNA damage.

    Science.gov (United States)

    Chung, Long H; Murray, Vincent

    2018-01-01

    The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2005-06-01

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

  8. Photorepair of UV damage to DNA: purification and properties of DNA photolyase (the DNA-photoreactivating enzyme). Progress report, August 1, 1975--July 31, 1976

    International Nuclear Information System (INIS)

    Werbin, H.

    1976-01-01

    Progress is reported on the following research projects: separation of photolyase subunits by sucrose gradient sedimentation; determination of whether fluorescent material is the chromophore for photolyase; studies on tryptophane and lysine residues to determine whether these are involved in the binding and photolytic steps; nmr spectrum of activator of photolyase; damage to pea chromatin by solar near uv and repair of damage; tryptophan residues in yeast DNA photolyase; photolyase in pea seedlings; and nuclear magnetic resonance spectra of purified activator

  9. A Fluorescent Probe to Measure DNA Damage and Repair.

    Directory of Open Access Journals (Sweden)

    Allison G Condie

    Full Text Available DNA damage and repair is a fundamental process that plays an important role in cancer treatment. Base excision repair (BER is a major repair pathway that often leads to drug resistance in DNA-targeted cancer chemotherapy. In order to measure BER, we have developed a near infrared (NIR fluorescent probe. This probe binds to a key intermediate, termed apurinic/apyrimidinic (AP site, in the BER pathway where DNA damage and repair occurs. We have developed an assay to show the efficacy of the probe binding to AP sites and have shown that it can distinguish AP sites in DNA extract from chemotherapy treated cells. This probe has potential application in monitoring patient response to chemotherapy and evaluating new drugs in development.

  10. Harnessing the p53-PUMA Axis to Overcome DNA Damage Resistance in Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Xiaoguang Zhou

    2014-12-01

    Full Text Available Resistance to DNA damage–induced apoptosis is a hallmark of cancer and a major cause of treatment failure and lethal disease outcome. A tumor entity that is largely resistant to DNA-damaging therapies including chemo- or radiotherapy is renal cell carcinoma (RCC. This study was designed to explore the underlying molecular mechanisms of DNA damage resistance in RCC to develop strategies to resensitize tumor cells to DNA damage–induced apoptosis. Here, we show that apoptosis-resistant RCC cells have a disconnect between activation of p53 and upregulation of the downstream proapoptotic protein p53 upregulated modulator of apoptosis (PUMA. We demonstrate that this disconnect is not caused by gene-specific repression through CCCTC-binding factor (CTCF but instead by aberrant chromatin compaction. Treatment with an HDAC inhibitor was found to effectively reactivate PUMA expression on the mRNA and protein level and to revert resistance to DNA damage–induced cell death. Ectopic expression of PUMA was found to resensitize a panel of RCC cell lines to four different DNA-damaging agents tested. Remarkably, all RCC cell lines analyzed were wild-type for p53, and a knockdown was likewise able to sensitize RCC cells to acute genotoxic stress. Taken together, our results indicate that DNA damage resistance in RCC is reversible, involves the p53-PUMA axis, and is potentially targetable to improve the oncological outcomes of RCC patients.

  11. DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

    Directory of Open Access Journals (Sweden)

    Monika Hlavová

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

  12. Linking abnormal mitosis to the acquisition of DNA damage

    Science.gov (United States)

    Pellman, David

    2012-01-01

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

  13. DNA damage and repair in human skin in situ

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-01

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

  14. The current state of eukaryotic DNA base damage and repair.

    Science.gov (United States)

    Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

    2015-12-02

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Statistical analysis of post mortem DNA damage-derived miscoding lesions in Neandertal mitochondrial DNA

    DEFF Research Database (Denmark)

    Vives, Sergi; Gilbert, M Thomas; Arenas, Conchita

    2008-01-01

    ABSTRACT: BACKGROUND: We have analysed the distribution of post mortem DNA damage derived miscoding lesions from the datasets of seven published Neandertal specimens that have extensive cloned sequence coverage over the mitochondrial DNA (mtDNA) hypervariable region 1 (HVS1). The analysis......-->A miscoding lesions (observed ratio of 67:2 compared to an expected ratio of 7:2), implying that the mtDNA Light strand molecule suffers proportionally more damage-derived miscoding lesions than the Heavy strand. CONCLUSION: The clustering of Cs in the Light strand as opposed to the singleton pattern of Cs...

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

    Directory of Open Access Journals (Sweden)

    Hamidreza Namazi

    2015-01-01

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

  17. Biomarkers of oxidative damage to DNA and repair

    DEFF Research Database (Denmark)

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone

    2008-01-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation......,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine......, which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7...

  18. A Mathematical Model for DNA Damage and Repair

    Directory of Open Access Journals (Sweden)

    Philip S. Crooke

    2010-01-01

    Full Text Available In cells, DNA repair has to keep up with DNA damage to maintain the integrity of the genome and prevent mutagenesis and carcinogenesis. While the importance of both DNA damage and repair is clear, the impact of imbalances between both processes has not been studied. In this paper, we created a combined mathematical model for the formation of DNA adducts from oxidative estrogen metabolism followed by base excision repair (BER of these adducts. The model encompasses a set of differential equations representing the sequence of enzymatic reactions in both damage and repair pathways. By combining both pathways, we can simulate the overall process by starting from a given time-dependent concentration of 17β-estradiol (E2 and 2′-deoxyguanosine, determine the extent of adduct formation and the correction by BER required to preserve the integrity of DNA. The model allows us to examine the effect of phenotypic and genotypic factors such as different concentrations of estrogen and variant enzyme haplotypes on the formation and repair of DNA adducts.

  19. The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage

    DEFF Research Database (Denmark)

    Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A

    2014-01-01

    Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recrui...

  20. BRCA1 and BRCA2 heterozygosity and repair of X-ray-induced DNA damage

    NARCIS (Netherlands)

    Nieuwenhuis, B.; Van Assen-Bolt, AJ; van Waarde-Verhagen, Maria; Sijmons, R.J.; van der Hout, A.H.; Bauch, T; Streffer, C; Kampinga, H.H.

    Purpose: Up to 90% of hereditary breast cancer cases are linked to germ-line mutations in one of the two copies of the BRCA1 or BRCA2 genes. Brca1 and Brca2 proteins are both involved in the cellular defence against DNA damage, although the precise function of the proteins is still not known. Some

  1. Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response

    DEFF Research Database (Denmark)

    Fazio, Alessandro

    to investigate the transcriptional and phenotypic responses to the alkylating agent methyl methanesulfonate (MMS) of mutant strains carrying deletions of genes encoding protein kinases (Mec1, Tel1, Rad53, Dun1, Chk1, Alk1) and protein phosphatases (Ptc3, Pph3, Oca1) involved in DNA damage response (DDR). We have...

  2. Heterochromatin protein 1 is recruited to various types of DNA damage

    NARCIS (Netherlands)

    Luijsterburg, Martijn S.; Dinant, Christoffel; Lans, Hannes; Stap, Jan; Wiernasz, Elzbieta; Lagerwerf, Saskia; Warmerdam, Daniël O.; Lindh, Michael; Brink, Maartje C.; Dobrucki, Jurek W.; Aten, Jacob A.; Fousteri, Maria I.; Jansen, Gert; Dantuma, Nico P.; Vermeulen, Wim; Mullenders, Leon H. F.; Houtsmuller, Adriaan B.; Verschure, Pernette J.; van Driel, Roel

    2009-01-01

    Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-alpha, HP1-beta, and HP1-gamma are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in

  3. Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA

    International Nuclear Information System (INIS)

    Bodell, W.J.; Cleaver, J.E.; Roti Roti, J.L.

    1984-01-01

    The authors have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 0 C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 0 C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus, the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating steps(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 0 C with[ 3 H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 0 C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures. 23 references, 3 figures, 2 tables

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

    Science.gov (United States)

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

    2014-01-01

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

  5. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    Science.gov (United States)

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

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  6. Effects of chemical-induced DNA damage on male germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Holme, J.A.; Bjoerge, C.; Trbojevic, M.; Olsen, A.K.; Brunborg, G.; Soederlund, E.J. [National Inst. of Public Health, Oslo (Norway). Dept. of Environmental Medicine; Bjoeras, M.; Seeberg, E. [National Hospital, Oslo (Norway). Dept. of Microbiology; Scholz, T.; Dybing, E.; Wiger, R. [National Hospital, Oslo (Norway). Inst. for Surgical Research and Surgical Dept. B

    1998-12-31

    Several recent studies indicate declines in sperm production, as well as increases in the incidence of genitourinary abnormalities such as testicular cancer, cryptorchidism and hypospadias. It is not known if these effects are due to exposure to chemical pollutants or if other ethiological factors are involved. Animal studies indicate that chemicals will induce such effects by various genetic, epigenetic or non-genetic mechanisms. Recently, much attention has been focused on embryonic/fetal exposure to oestrogen-mimicking chemicals (Toppari et al., 1996). However, the possibility that chemicals may cause reproductive toxicity by other mechanisms such as interactions with DNA, should not be ignored. DNA damage in germ cells may lead to the production of mutated spermatozoa, which in turn may result in spontaneous abortions, malformations and/or genetic defects in the offspring. Regarding the consequences of DNA alterations for carcinogenesis it is possible that genetic damage may occur germ cells, but the consequences are not expressed until certain genetic events occur in postnatal life. Transmission of genetic risk is best demonstrated by cancer-prone disorders such as hereditary retinoblastoma and the Li-Fraumeni syndrome. A number of experiments indicate that germ cells and proliferating cells may be particularly sensitive to DNA damaging agents compared to other cells. Furthermore, several lines of evidence have indicated that one of the best documented male reproductive toxicants, 1,2-dibrome-3-chloropropane (DBCP), causes testicular toxicity through DNA damage. It is possible that testicular cells at certain maturational stages are more subject to DNA damage, have less efficient DNA repair, or have different thresholds for initiating apoptosis following DNA damage than other cell types. (orig.)

  7. DNA damage: beta zero versus beta plus thalassemia.

    Science.gov (United States)

    Sagar, Chandan S; Kumar, Rakesh; Sharma, Dharmesh C; Kishor, Purnima

    2015-01-01

    β thalassemia results in an increase in the α to non-α chain ratio. Iron released from unpaired α chains in RBCs and that ensuing from regular transfusions is the major cause of cellular damage. The use of iron chelators to counter the iron overload is accompanied by side-effects. The extent of iron toxicity could vary from one patient to another and could help in determining the optimal chelator dose for each patient. To observe the pro-oxidant/antioxidant disturbance and the extent of DNA damage in β thalassemia patients with different β globin gene anomalies. The formation of Reactive Oxygen Species (ROS ) was observed by incubation of cell suspensions with 2',7', dichlorofluorescin-diacetate (DCFH DA) and DNA damage was demonstrated by single cell gel electrophoresis. Heinz bodies were observed by staining blood smears. The study group comprised 50 regularly transfused beta thalassemia patients and 40 non thalassemic controls. While Heinz bodies and nucleated RBCs were seen in all the patients, oxidation of DCFH and DNA damage were seen to be associated with the β globin gene defect. DNA damage was found to be greater in β(0) homozygotes as compared to the β(+) homozygotes, and was maximum in patients presenting with the 619 base pair deletion. In the present study, iron toxicity, as indicated by DNA damage, has been seen to vary in the patients. Thus, monitoring of the dose of iron chelators, according to the type of mutation in the beta globin gene, may help improve the compliance of beta thalassemics to chelation therapy and prevent side-effects in patients with beta plus mutations.

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

    Science.gov (United States)

    Hájková, Andrea; Barek, Jiří; Vyskočil, Vlastimil

    2017-08-01

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

  9. Repair of DNA damage in light sensitive human skin diseases

    Energy Technology Data Exchange (ETDEWEB)

    Horkay, I.; Varga, L.; Tam' asi P., Gundy, S.

    1978-12-01

    Repair of uv-light induced DNA damage and changes in the semiconservative DNA synthesis were studied by in vitro autoradiography in the skin of patients with lightdermatoses (polymorphous light eruption, porphyria cutanea tarda, erythropoietic protoporphyria) and xeroderma pigmentosum as well as in that of healthy controls. In polymorphous light eruption the semiconservative DNA replication rate was more intensive in the area of the skin lesions and in the repeated phototest site, the excision repair synthesis appeared to be unaltered. In cutaneous prophyrias a decreased rate of the repair incorporation could be detected. Xeroderma pigmentosum was characterized by a strongly reduced repair synthesis.

  10. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

    2014-08-05

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting.

  11. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    International Nuclear Information System (INIS)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

    2014-01-01

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting

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

    Science.gov (United States)

    Luftig, Micah A

    2014-11-01

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

  13. Repair Mechanism of UV-damaged DNA in Xeroderma Pigmentosum | Center for Cancer Research

    Science.gov (United States)

    Xeroderma pigmentosum (XP) is a rare, inherited disorder characterized by extreme skin sensitivity to ultraviolet (UV) rays from sunlight. XP is caused by mutations in genes involved in nucleotide excision repair (NER) of damaged DNA. Normal cells are usually able to fix this damage before it leads to problems; however, the DNA damage is not repaired normally in patients with XP. As more abnormalities form in DNA, cells malfunction and eventually become cancerous or die. XP patients have more than a 10,000-fold increased risk of developing skin cancer. Kenneth Kraemer, M.D., in CCR’s Dermatology Branch, has been studying XP patients at the Clinical Center for more than 40 years.

  14. Rho GTPases: Novel Players in the Regulation of the DNA Damage Response?

    Directory of Open Access Journals (Sweden)

    Gerhard Fritz

    2015-09-01

    Full Text Available The Ras-related C3 botulinum toxin substrate 1 (Rac1 belongs to the family of Ras-homologous small GTPases. It is well characterized as a membrane-bound signal transducing molecule that is involved in the regulation of cell motility and adhesion as well as cell cycle progression, mitosis, cell death and gene expression. Rac1 also adjusts cellular responses to genotoxic stress by regulating the activity of stress kinases, including c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK and p38 kinases as well as related transcription factors. Apart from being found on the inner side of the outer cell membrane and in the cytosol, Rac1 has also been detected inside the nucleus. Different lines of evidence indicate that genotoxin-induced DNA damage is able to activate nuclear Rac1. The exact mechanisms involved and the biological consequences, however, are unclear. The data available so far indicate that Rac1 might integrate DNA damage independent and DNA damage dependent cellular stress responses following genotoxin treatment, thereby coordinating mechanisms of the DNA damage response (DDR that are related to DNA repair, survival and cell death.

  15. Evaluation of the DNA damaging effects of amitraz on human ...

    Indian Academy of Sciences (India)

    2013-01-05

    Jan 5, 2013 ... Agriculture, 3Department of Microbiology, Faculty of Biology, The University of Belgrade,. Belgrade, Serbia ... important uses against ticks, mites and lice on animals. Also, amitraz is used ... It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes.

  16. Dissection of DNA damage responses using multiconditional genetic interaction maps

    NARCIS (Netherlands)

    Guénolé, Aude

    2013-01-01

    To protect the genome, cells have evolved a diverse set of pathways designed to sense, signal, and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among these pathways, we systematically mapped changes in the cell's genetic network across a panel of different

  17. Repair of ultraviolet damage in Haemophilus influenzae DNA

    International Nuclear Information System (INIS)

    Setlow, J.K.; LeClerc, J.E.

    1975-01-01

    Excision and postreplication repair in Haemophilus influenzae differ in a number of respects from these well-known repair processes in Escherichia coli. Excision-repair of transforming DNA takes place only after its integration. Like other readily transformable bacteria, Haemophilus influenzae does not contain any photoreactivating enzyme. UV damage in this microorganism is repaired by an excision mechanism and by postreplication repair

  18. DNA damage and plasma homocysteine levels are associated with ...

    African Journals Online (AJOL)

    This study describes the association between levels of DNA damage and homocysteine (Hcy) in persistent diarrheic (PD) patients and correlates them with serum biochemical metabolites and mineral components. PD patients (n = 36) age 4 - 6 years from Faisalabad hospitals were examined for anthropometric factors, ...

  19. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    Science.gov (United States)

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

  20. DNA damage and plasma homocysteine levels are associated with ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-01-18

    Jan 18, 2010 ... This study describes the association between levels of DNA damage and homocysteine (Hcy) in persistent diarrheic (PD) patients and correlates them with serum biochemical metabolites and mineral components. PD patients (n = 36) age 4 - 6 years from Faisalabad hospitals were examined for.

  1. UV Radiation Damage and Bacterial DNA Repair Systems

    Science.gov (United States)

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

    2006-01-01

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

  2. The DNA-damage response in human biology and disease

    Czech Academy of Sciences Publication Activity Database

    Jackson, S.P.; Bartek, Jiří

    2009-01-01

    Roč. 461, č. 7267 (2009), s. 1071-1078 ISSN 0028-0836 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA damage response * human disease * cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 34.480, year: 2009

  3. SUMO boosts the DNA damage response barrier against cancer

    Czech Academy of Sciences Publication Activity Database

    Bartek, Jiří; Hodný, Zdeněk

    2010-01-01

    Roč. 17, č. 1 (2010), s. 9-11 ISSN 1535-6108 R&D Projects: GA ČR GA301/08/0353 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA damage response * ubiquitylation * sumoylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 26.925, year: 2010

  4. Oxidatively damaged DNA in animals exposed to particles

    DEFF Research Database (Denmark)

    Møller, Peter; Danielsen, Pernille Høgh; Jantzen, Kim

    2013-01-01

    from animal experimental models that both pulmonary and gastrointestinal tract exposure to particles are associated with elevated levels of oxidatively damaged DNA in the lung and internal organs. However, there is a paucity of studies on pulmonary exposure to low doses of particles that are relevant...

  5. The DNA damage response: The omics era and its impact

    NARCIS (Netherlands)

    K.W.J. Derks (Kasper); J.H.J. Hoeijmakers (Jan); J. Pothof (Joris)

    2014-01-01

    textabstractThe 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,

  6. The DNA-damage response in human biology and disease

    DEFF Research Database (Denmark)

    Jackson, Stephen P; Bartek, Jiri

    2009-01-01

    , signal its presence and mediate its repair. Such responses, which have an impact on 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....

  7. Systemic oxidatively generated DNA/RNA damage in clinical depression

    DEFF Research Database (Denmark)

    Jorgensen, Anders; Krogh, Jesper; Miskowiak, Kamilla

    2013-01-01

    oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy...

  8. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    2015-02-04

    Feb 4, 2015 ... DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer. [Mittra I, Khare NK, Raghuram GV, Chaubal R, Khambatti F, Gupta D, Gaikwad A, Prasannan P, Singh A, Iyer A, Singh A ...

  9. Evaluation of the DNA damaging effects of amitraz on human ...

    Indian Academy of Sciences (India)

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz ...

  10. Detecting DNA damage with a silver solid amalgam electrode

    Czech Academy of Sciences Publication Activity Database

    Kuchaříková, Kateřina; Novotný, Ladislav; Josypčuk, Bohdan; Fojta, Miroslav

    2004-01-01

    Roč. 16, č. 5 (2004), s. 410-414 ISSN 1040-0397 R&D Projects: GA AV ČR IAA4004108; GA AV ČR IBS5004355 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA damage * silver solid amalgam electrode * HMDE Subject RIV: BO - Biophysics Impact factor: 2.038, year: 2004

  11. Role of DNA repair in repair of cytogenetic damages. Contribution of repair of single-strand DNA breaks to cytogenetic damages repair

    International Nuclear Information System (INIS)

    Rozanova, O.M.; Zaichkina, S.I.; Aptikaev, G.F.; Ganassi, E.Eh.

    1989-01-01

    The comparison was made between the results of the effect of poly(ADP-ribosylation) ingibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase ingibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronuclear test, and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modyfying effect with regard to radiation cytogenetic damages and kynetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes

  12. Micronutrients intake associated with DNA damage assessed by in a human biomonitoring study

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-05-01

    Retinol was positively correlated with oxidative DNA damage in controls. The study by van Helden et al. (2009 demonstrated that vitamin A enhances OH radical formation in the Fenton reaction, showing that vitamin A can act as pro-oxidant or antioxidant, depending on the type of radicals involved, and may lead to DNA oxidative damage (Alakhras et al., 2011. Azqueta & Collins (2012 clearly distinguished between effects of vitamin A, pro-vitamin A carotenoids, and non-vitamin A carotenoids; being the latter group almost invariably reported to protect against DNA damage, whether endogenous or induced by exogenous agents, the pro-vitamin A carotenoids show a wider spectrum of effects, sometimes protecting and sometimes enhancing DNA damage. Vitamin E was found to be positively correlated with % DNA in tail. Watters et al. (2007 also found a positive association of vitamin E and oxidative DNA damage in a healthy, non-smoking population of young adults. A possible explanation for this result stems from some evidence that in the presence of copper or in smokers with a fat rich diet, vitamin E can act as a strong pro-oxidant, nevertheless it remains an unexpected result. Results found a positive correlation between iron and % DNA in tail, meaning that higher intake of iron associates with higher DNA damage. Oxidative lesions, and more specifically 8-OHdG, is one of the most prevalent lesions induced by iron containing substances (Prá et al., 2012, however the FPG biomarker was not statistically associated with iron. There is sound evidence that iron deficiency increases genome instability, among other mechanisms, by impairing enzymes involved in antioxidant and nuclei acid metabolism (Prá et al., 2012. Results presented herein found that the amount of calories ingested was negatively correlated with both biomarkers assessed by comet assay. This was somewhat unexpected, as calories restriction reduces metabolic rate and oxidative stress, meaning that lower calories

  13. Radiation damage to DNA: The importance of track structure

    CERN Document Server

    Hill, M A

    1999-01-01

    A wide variety of biological effects are induced by ionizing radiation, from cell death to mutations and carcinogenesis. The biological effectiveness is found to vary not only with the absorbed dose but also with the type of radiation and its energy, i.e., with the nature of radiation tracks. An overview is presented of some of the biological experiments using different qualities of radiation, which when compared with Monte Carlo track structure studies, have highlighted the importance of the localized spatial properties of stochastic energy deposition on the nanometer scale at or near DNA. The track structure leads to clustering of damage which may include DNA breaks, base damage etc., the complexity of the cluster and therefore its biological repairability varying with radiation type. The ability of individual tracks to produce clustered damage, and the subsequent biological response are important in the assessment of the risk associated with low-level human exposure. Recent experiments have also shown that...

  14. DNA damage and repair efficiency in lymphocytes from schizophrenic patients.

    Science.gov (United States)

    Psimadas, Dimitrios; Messini-Nikolaki, Niki; Zafiropoulou, Maria; Fortos, Andreas; Tsilimigaki, Smaragdi; Piperakis, Stylianos M

    2004-02-10

    In the present study we examined schizophrenic patients' lymphocytes sensitivity to the effects of external factors, such as hydrogen peroxide and gamma-irradiation and also their repair efficiency with the comet assay. Our results did no show any difference in basal levels of DNA damage between schizophrenic and normal populations. The slightly increased sensitivity of the schizophrenic population to the externally induced DNA damage compared to controls was not statistically significant. Also the small reduction in the DNA repair efficiency in schizophrenics in comparison to normal population was found to be not statistically significant. Finally, patients with heritable predisposition to schizophrenia did not show any difference in their response from the other schizophrenics.

  15. Oxidatively generated DNA/RNA damage in psychological stress states

    DEFF Research Database (Denmark)

    Jørgensen, Anders

    2013-01-01

    Both non-pathological psychological stress states and mental disorders are associated with molecular, cellular and epidemiological signs of accelerated aging. Oxidative stress on nucleic acids is a critical component of cellular and organismal aging, and a suggested pathogenic mechanism in several...... age-related somatic disorders. The overall aim of the PhD project was to investigate the relation between psychopathology, psychological stress, stress hormone secretion and oxidatively generated DNA and RNA damage, as measured by the urinary excretion of markers of whole-body DNA/RNA oxidation (8......-oxodG and 8-oxoGuo, respectively). The main hypothesis was that psychological stress states are associated with increased DNA/RNA damage from oxidation. In a study of 40 schizophrenia patients and 40 healthy controls matched for age and gender, we found that 8-oxodG/8-oxoGuo excretion was increased...

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

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Lorenzo, Y.; Karlsen, A.; Carlsen, M. H.; Novosadová, Vendula; Blomhoff, R.; Vodička, Pavel; Collins, A. R.

    2014-01-01

    Roč. 16, APR 2014 (2014), s. 66-73 ISSN 1568-7864 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : DNA damage * DNA repair capacity * diet Subject RIV: EB - Genetics ; Molecular Biology; EI - Biotechnology ; Bionics (BTO-N) Impact factor: 3.111, year: 2014

  17. Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans

    DEFF Research Database (Denmark)

    Møller, P; Loft, S; Lundby, C

    2001-01-01

    The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test...... exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive...... oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity...

  18. Telomeric Allelic Imbalance Indicates Defective DNA Repair and Sensitivity to DNA-Damaging Agents

    DEFF Research Database (Denmark)

    Birkbak, Nicolai J.; Wang, Zhigang C.; Kim, Ji-Young

    2012-01-01

    also benefit from these agents. NtAI, a genomic measure of unfaithfully repaired DNA, may identify cancer patients likely to benefit from treatments targeting defective DNA repair. Cancer Discov; 2(4); 366–75. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 288......DNA repair competency is one determinant of sensitivity to certain chemotherapy drugs, such as cisplatin. Cancer cells with intact DNA repair can avoid the accumulation of genome damage during growth and also can repair platinum-induced DNA damage. We sought genomic signatures indicative...... of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (NtAI) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients...

  19. Oxidative damage of DNA in subjects occupationally exposed to lead.

    Science.gov (United States)

    Pawlas, Natalia; Olewińska, Elżbieta; Markiewicz-Górka, Iwona; Kozłowska, Agnieszka; Januszewska, Lidia; Lundh, Thomas; Januszewska, Ewa; Pawlas, Krystyna

    2017-09-01

    Exposure to lead (Pb) in environmental and occupational settings continues to be a serious public health problem and may pose an elevated risk of genetic damage. The aim of this study was to assess the level of oxidative stress and DNA damage in subjects occupationally exposed to lead. We studied a population of 78 male workers exposed to lead in a lead and zinc smelter and battery recycling plant and 38 men from a control group. Blood lead levels were detected by graphite furnace atomic absorption spectrophotometry and plasma lead levels by inductively coupled plasma-mass spectrometry. The following assays were performed to assess the DNA damage and oxidative stress: comet assay, determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG), lipid peroxidation and total antioxidant status (TAS). The mean concentration of lead in the blood of the exposed group was 392 ± 103 μg/L and was significantly higher than in the control group (30.3 ± 29.4 μg/L, p lead exposure [lead in blood, lead in plasma, zinc protoporphyrin (ZPP)] and urine concentration of 8-OHdG. The level of oxidative damage of DNA was positively correlated with the level of lipid peroxidation (TBARS) and negatively with total anti-oxidative status (TAS). Our study suggests that occupational exposure causes an increase in oxidative damage to DNA, even in subjects with relatively short length of service (average length of about 10 years). 8-OHdG concentration in the urine proved to be a sensitive and non-invasive marker of lead induced genotoxic damage.

  20. The chemical basis of DNA damage by the direct pathway of ionizing radiation

    International Nuclear Information System (INIS)

    Sharma, Kiran Kumar K.

    2013-01-01

    Free radicals in living system has been implicated as playing a major role in the etiology of variety of diseases. The mechanism of free radicals in vivo involves predominantly the reaction with the DNA, producing different types of damage to the DNA. These lesions induced to the DNA could lead to mutation and even cell death. Radiolysis techniques, which uses ionizing radiation has proven to be one of the most advanced and excellent tool for studying the free radical reaction mechanisms as it can produce a host of well characterized free radicals. The effects of ionizing radiation on DNA have been studied for many years. Ionizing radiation interacts with DNA in vivo by two pathways, direct and indirect. The indirect accounts for 50-60% while the direct effect accounts for 40-50%. The chemical mechanism of the former reaction arising mainly from the reactive species produced by radiolysis of water has been extensively studied, however with respect to the later pathway, which creates holes and electrons to the DNA molecule using DNA films and crystals is an active area of research as both the pathways plays important roles in DNA damage in vivo particularly in chromosomal DNA which are tightly bound with histones and compartmentalized

  1. Linking loss of sodium-iodide symporter expression to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Lyckesvärd, Madeleine Nordén [Sahlgrenska Cancer Center, University of Gothenburg, Göteborg (Sweden); Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Kapoor, Nirmal [Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Ingeson-Carlsson, Camilla; Carlsson, Therese [Sahlgrenska Cancer Center, University of Gothenburg, Göteborg (Sweden); Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Karlsson, Jan-Olof [Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Postgård, Per; Himmelman, Jakob; Forssell-Aronsson, Eva [Department of Radiation Physics, University of Gothenburg, Göteborg (Sweden); Hammarsten, Ola [Department of Clinical Chemistry, University of Gothenburg, Göteborg (Sweden); Nilsson, Mikael, E-mail: mikael.nilsson@gu.se [Sahlgrenska Cancer Center, University of Gothenburg, Göteborg (Sweden); Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden)

    2016-05-15

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

  2. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    OpenAIRE

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

    2014-01-01

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be import...

  3. Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

    OpenAIRE

    Korycka-Machala, M; Brzostek, A; Rozalska, S; Rumijowska-Galewicz, A; Dziedzic, R; Bowater, R; Dziadek, J

    2006-01-01

    Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the respon...

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

    Directory of Open Access Journals (Sweden)

    Nadine Nilles

    2017-05-01

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

  5. Measurement of oxidatively generated base damage in cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, Jean, E-mail: jean.cadet@cea.fr [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no3 (CEA/UJF), FRE CNRS 3200, Departement de Recherche Fondamentale sur la Matiere Condensee, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France); Douki, Thierry; Ravanat, Jean-Luc [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no3 (CEA/UJF), FRE CNRS 3200, Departement de Recherche Fondamentale sur la Matiere Condensee, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France)

    2011-06-03

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

  6. New Modeling Approaches to Study DNA Damage by the Direct and Indirect Effects of Ionizing Radiation

    Science.gov (United States)

    Plante, Ianik; Cucinotta, Francis A.

    2012-01-01

    DNA is damaged both by the direct and indirect effects of radiation. In the direct effect, the DNA itself is ionized, whereas the indirect effect involves the radiolysis of the water molecules surrounding the DNA and the subsequent reaction of the DNA with radical products. While this problem has been studied for many years, many unknowns still exist. To study this problem, we have developed the computer code RITRACKS [1], which simulates the radiation track structure for heavy ions and electrons, calculating all energy deposition events and the coordinates of all species produced by the water radiolysis. In this work, we plan to simulate DNA damage by using the crystal structure of a nucleosome and calculations performed by RITRACKS. The energy deposition events are used to calculate the dose deposited in nanovolumes [2] and therefore can be used to simulate the direct effect of the radiation. Using the positions of the radiolytic species with a radiation chemistry code [3] it will be possible to simulate DNA damage by indirect effect. The simulation results can be compared with results from previous calculations such as the frequencies of simple and complex strand breaks [4] and with newer experimental data using surrogate markers of DNA double ]strand breaks such as . ]H2AX foci [5].

  7. DNA repair in human cells: Methods for the determination of calmodulin involvement

    International Nuclear Information System (INIS)

    Charp, P.A.

    1987-01-01

    Exposure of DNA to either physical or chemical agents can result in the formation of a number of different lesions which must be repaired enzymatically in order for DNA to carry on normal replication and transcription. In most cases, the enzymes involved in this repair of damaged DNA include endonucleases, exonucleases, glycosylases, polymerases, and ligases. Each group of enzymes is involved in precise steps in DNA repair. Exposure to physical agents such as ultraviolet light (UV) at a wavelength of 254 nm is repaired by two distinct and different mechanisms. One mode of enzymatic repair of pyrimidine dimers is accomplished in situ by photoreactivation of UV-induced pyrimidine dimers by photoreactivating light. The second mode of enzymatic repair is the excision repair of pyrimidine dimers involving several different enzymes including endonuclease, exonuclease, and DNA ligase. A summary of the sequence of enzymatic steps involved is shown. It has been observed that specific drugs which bind to and alter the action of calmodulin in cells block DNA synthesis. This suggests that calmodulin may play a role both in normal DNA replication and repair. Others using an indirect method measuring the degree of DNA nucleoid sedimentation, showed that the specific anti-calmodulin agent W-13 slowed the rate of DNA repair. Others showed that DNA synthesis in T51B rat liver cells could be blocked with the addition of either chlorpromazine or trifluoperazine

  8. Dynamic changes to survivin subcellular localization are initiated by DNA damage

    Directory of Open Access Journals (Sweden)

    Maritess Gay Asumen

    2010-07-01

    Full Text Available Maritess Gay Asumen1, Tochukwu V Ifeacho2, Luke Cockerham3, Christina Pfandl4, Nathan R Wall31Touro University’s College of Osteopathic Medicine, Vallejo, CA, USA; 2University of Southern California, Los Angeles, CA, USA; 3Center for Health Disparities Research and Molecular Medicine, Loma Linda University, CA, USA; 4Green Mountain Antibodies, Burlington, VT, USAAbstract: Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light- initiated DNA damage and that its distribution may be responsible for its multifunctionality.Keywords: survivin, PIK kinases, ATM, ATR, DNA-PK

  9. Endogenous melatonin and oxidatively damaged guanine in DNA

    Directory of Open Access Journals (Sweden)

    Poulsen Henrik E

    2009-10-01

    Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

  10. Repair of DNA damage in the human metallothionein gene family

    International Nuclear Information System (INIS)

    Leadon, S.A.; Snowden, M.M.

    1987-01-01

    In order to distinguish enhanced repair of a sequence due to its transcriptional activity from enhanced repair due to chromatin alterations brought about by integration of a sequence into the genome, we have investigated the repair of damage both in endogenous genes and in cell lines that contain an integrated gene with an inducible promoter. The endogenous genes we are studying are the metallothioneins (MTs), a multigene family in man consisting of about 10-12 members. Cultured cells were exposed to 10-J/m 2 uv light and allowed to repair in the presence of bromodeoxyuridine. The DNA was then isolated, digested with Eco RI, and fully hybrid density DNA made by semiconservative synthesis was separated from unreplicated DNA by centrifugation in CsCl density gradients. Unreplicated, parental-density DNA was then reacted with a monoclonal antibody against bromouracil. 1 ref., 1 fig., 1 tab

  11. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

    Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity...... with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation...... of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

  12. Reconstitution of UV-damaged DNA into chromatin using Xenopus oocyte extracts

    International Nuclear Information System (INIS)

    Widlak, P.

    1998-01-01

    Chromatin was reconstituted in vitro using Xenopus oocyte extracts and plasmid DNA containing UV radiation-induced damage. Damaged DNA was assembled into minichromosomes with an efficiency similar to that of control, non-irradiated DNA. Oocyte extract s were competent to carry out DNA repair, which was elicited by nicking damaged templates followed by DNA synthesis during chromatin assembly. Newly synthesized DNA was efficiently reconstituted into nucleosomes. (author)

  13. Damage-induced DNA repair processes in Escherichia coli cells

    International Nuclear Information System (INIS)

    Slezarikova, V.

    1986-01-01

    The existing knowledge is summed up of the response of Escherichia coli cells to DNA damage due to various factors including ultraviolet radiation. So far, three inducible mechanisms caused by DNA damage are known, viz., SOS induction, adaptation and thermal shock induction. Greatest attention is devoted to SOS induction. Its mechanism is described and the importance of the lexA recA proteins is shown. In addition, direct or indirect role is played by other proteins, such as the ssb protein binding the single-strand DNA sections. The results are reported of a study of induced repair processes in Escherichia coli cells repeatedly irradiated with UV radiation. A model of induction by repeated cell irradiation discovered a new role of induced proteins, i.e., the elimination of alkali-labile points in the daughter DNA synthetized on a damaged model. The nature of the alkali-labile points has so far been unclear. In the adaptation process, regulation proteins are synthetized whose production is induced by the presence of alkylation agents. In the thermal shock induction, new proteins synthetize in cells, whose function has not yet been clarified. (E.S.)

  14. Sensitive detection of DNA oxidation damage induced by nanomaterials.

    Science.gov (United States)

    Collins, Andrew; El Yamani, Naouale; Dusinska, Maria

    2017-06-01

    From a toxicological point of view, nanomaterials are of interest; because - on account of their great surface area relative to mass - they tend to be more reactive than the bulk chemicals from which they are derived. They might in some cases have the potential to damage DNA directly, or could act via the induction of oxidative stress. The comet assay (single cell gel electrophoresis) is widely used to measure DNA strand breaks and also oxidised bases, by including in the procedure digestion with lesion-specific enzymes such as formamidopyrimidine DNA glycosylase (which converts oxidised purines to breaks) or endonuclease III (recognising oxidised pyrimidines). We summarise reports in which these enzymes have been used to study a variety of nanomaterials in diverse cell types. We also stress that it is important to carry out tests of cell viability alongside the genotoxicity assay, since cytotoxicity can lead to adventitious DNA damage. Different concentrations of nanomaterials should be investigated, concentrating on a non-cytotoxic range; and incubating for short and longer periods can give valuable information about the mode of damage induction. The use of lesion-specific enzymes can substantially enhance the sensitivity of the comet assay in detecting genotoxic effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Reduction in oxidatively generated DNA damage following smoking cessation

    Directory of Open Access Journals (Sweden)

    Freund Harold G

    2011-05-01

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

  16. Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Johannessen, Christian; Rasmussen, Lene Juel

    2009-01-01

    proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little...... is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[def,mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures...... in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible...

  17. Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

    DEFF Research Database (Denmark)

    Loft, S; Poulsen, H E; Vistisen, K

    1999-01-01

    pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers...... with the 8-oxodG excretion. The increased excretion of 8-oxodG in bus drivers from central Copenhagen as compared with drivers from rural/suburban greater Copenhagen suggests that exposure to ambient air pollution causes oxidative damage to DNA. This effect may be modified by the activity of CYP1A2......Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air...

  18. Fructose-1,6-bisphosphatase mediates cellular responses to DNA damage and aging in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kitanovic, Ana; Woelfl, Stefan

    2006-01-01

    Response to DNA damage, lack of nutrients and other stress conditions is an essential property of living systems. The coordinate response includes DNA damage repair, activation of alternate biochemical pathways, adjustment of cellular proliferation and cell cycle progression as well as drastic measures like cellular suicide which prevents proliferation of severely damaged cells. Investigating the transcriptional response of Saccharomyces cerevisiae to low doses of the alkylating agent methylmethane sulfonate (MMS) we observed induction of genes involved in glucose metabolism. RT-PCR analysis showed that the expression of the key enzyme in gluconeogenesis fructose-1,6-bisphosphatase (FBP1) was clearly up-regulated by MMS in glucose-rich medium. Interestingly, deletion of FBP1 led to reduced sensitivity to MMS, but not to other DNA-damaging agents, such as 4-NQO or phleomycin. Reintroduction of FBP1 in the knockout restored the wild-type phenotype while overexpression increased MMS sensitivity of wild-type, shortened life span and increased induction of RNR2 after treatment with MMS. Deletion of FBP1 reduced production of reactive oxygen species (ROS) in response to MMS treatment and in untreated aged cells, and increased the amount of cells able to propagate and to form colonies, but had no influence on the genotoxic effect of MMS. Our results indicate that FBP1 influences the connection between DNA damage, aging and oxidative stress through either direct signalling or an intricate adaptation in energy metabolism

  19. Initiation of the ATM-Chk2 DNA damage response through the base excision repair pathway.

    Science.gov (United States)

    Chou, Wen-Cheng; Hu, Ling-Yueh; Hsiung, Chia-Ni; Shen, Chen-Yang

    2015-08-01

    The DNA damage response (DDR) is activated by various genotoxic stresses. Base lesions, which are structurally simple and predominantly fixed by base excision repair (BER), can trigger the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway, a DDR component. How these lesions trigger DDR remains unclear. Here we show that, for alkylation damage, methylpurine-DNA glycosylase (MPG) and apurinic/apyrimidinic endonuclease 1, both of which function early in BER, are required for ATM-Chk2-dependent DDR. In addition, other DNA glycosylases, including uracil-DNA glycosylase and 8-oxoguanine glycosylase, which are involved in repairing deaminated bases and oxidative damage, also induced DDR. The early steps of BER therefore play a vital role in modulating the ATM-Chk2 DDR in response to base lesions, facilitating downstream BER processing for repair, in which the formation of a single-strand break was shown to play a critical role. Moreover, MPG knockdown rescued cell lethality, its overexpression led to cell death triggered by DNA damage and, more interestingly, higher MPG expression in breast and ovarian cancers corresponded with a greater probability of relapse-free survival after chemotherapy, underscoring the importance of glycosylase-dependent DDR. This study highlights the crosstalk between BER and DDR that contributes to maintaining genomic integrity and may have clinical applications in cancer therapy. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Hispidin produced from Phellinus linteus protects against peroxynitrite-mediated DNA damage and hydroxyl radical generation.

    Science.gov (United States)

    Chen, Wei; Feng, Lina; Huang, Zhaoyi; Su, Hongming

    2012-09-30

    Oxidative stress plays an important role in the progression of many chronic diseases including cardiovascular diseases, diabetes, cancer and neurodegenerative disorders. One such mediator of oxidative stress is peroxynitrite, which is highly toxic to cultured neurons and astrocytes, and has been reported to be involved in the pathogenesis of various types of neuronal diseases. Therefore, searching for natural compounds with peroxynitrite-scavenging activity might be an effective therapy for peroxynitrite-mediated cytotoxicity. Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong antioxidant, anticancer, and antidiabetic properties. However, the astrocyte protective efficacy of hispidin has been not examined. This study was undertaken to investigate whether the astrocyte protective effect of hispidin is associated with inhibition of peroxynitrite-induced DNA damage, a critical event leading to peroxynitrite-mediated cytotoxicity. Our results showed that peroxynitrite can cause DNA damage in φX-174 plasmid DNA and rat primary astrocytes. The presence of hispidin (10-20 μg/ml) was found to significantly inhibit peroxynitrite-induced DNA damage and cytotoxicity. EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that hispidin potently diminished the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that hispidin can protect against peroxynitrite-mediated cytotoxicity, DNA damage and hydroxyl radical formation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Huselton, C.A.; Hill, H.Z. (New Jersey Medical School, Newark (USA))

    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.

  3. Characterization of ionizing radiation damage in DNA. Progress report, May 1, 1975--April 30, 1976

    International Nuclear Information System (INIS)

    Hawkins, R.B.

    1976-01-01

    The objective of this research is the characterization and quantitative assay of ionizing radiation-induced damage in DNA and nucleoprotein. Two lines of investigation have been pursued. The first is aimed at detection and assay of DNA to protein covalent cross linkage in coliphage T7. Protein and DNA are labeled with 14 C and 32 P, respectively. Cross linkage is assessed from the amount of labeled protein distributing like DNA and labeled DNA distributing like protein on a phenol-water countercurrent distribution system. It has been found that damage involving cross linkage occurs by two modes of radiation action in phage irradiated with 60 Co γ rays in .001M histidine medium. Indirect effects play a large role in one mode and direct effects play a large role in the other. In the second line of investigation, the hydrodynamic and viscoelastic properties of DNA from irradiated phage and cells will be examined to determine the extent to which DNA to DNA cross linkage and points of altered flexibility are introduced by radiation. An instrument for viscoelastic measurements has been constructed in preparation for these studies

  4. Damage to cellular and isolated DNA induced by a metabolite of aspirin

    Energy Technology Data Exchange (ETDEWEB)

    Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan)], E-mail: s-oikawa@doc.medic.mie-u.ac.jp; Kobayashi, Hatasu; Tada-Oikawa, Saeko [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); JSPS Research Fellow (Japan); Isono, Yoshiaki [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Kawanishi, Shosuke [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670 (Japan)

    2009-02-10

    Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H{sub 2}O{sub 2})-resistant clone HP100 cells, suggesting the involvement of H{sub 2}O{sub 2}. In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using {sup 32}P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H{sub 2}O{sub 2} with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

  5. Damage to cellular and isolated DNA induced by a metabolite of aspirin

    International Nuclear Information System (INIS)

    Oikawa, Shinji; Kobayashi, Hatasu; Tada-Oikawa, Saeko; Isono, Yoshiaki; Kawanishi, Shosuke

    2009-01-01

    Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H 2 O 2 )-resistant clone HP100 cells, suggesting the involvement of H 2 O 2 . In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using 32 P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H 2 O 2 with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis

  6. Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

    International Nuclear Information System (INIS)

    Meeusen, S.; Tieu, Q.; Wong, E.; Weiss, E.; Schieltz, D.; Yates, J.R.; Nunnari, J.

    1999-01-01

    Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome. (author)

  7. Preservation of ancient DNA in thermally damaged archaeological bone

    Science.gov (United States)

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

    2009-02-01

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

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

    Science.gov (United States)

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

    2009-02-01

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

  9. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    International Nuclear Information System (INIS)

    Pinto, A. Viviana; Deodato, Elder L.; Cardoso, Janine S.; Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K.; Leitao, Alvaro C.; Padula, Marcelo de

    2010-01-01

    Although titanium dioxide (TiO 2 ) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO 2 is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO 2 -UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO 2 associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO 2 plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO 2 protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO 2 plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO 2 plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  10. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, A. Viviana, E-mail: alicia.pinto@incqs.fiocruz.br [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Deodato, Elder L. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Cardoso, Janine S. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Leitao, Alvaro C. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Padula, Marcelo de [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil)

    2010-06-01

    Although titanium dioxide (TiO{sub 2}) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO{sub 2} is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO{sub 2}-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO{sub 2} associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO{sub 2} plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO{sub 2} protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO{sub 2} plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO{sub 2} plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  11. Protoparvovirus Interactions with the Cellular DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Kinjal Majumder

    2017-10-01

    Full Text Available Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM infects murine and transformed human cells provoking a sustained DNA damage response (DDR. This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.

  12. Protoparvovirus Interactions with the Cellular DNA Damage Response

    Science.gov (United States)

    Majumder, Kinjal; Etingov, Igor

    2017-01-01

    Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection. PMID:29088070

  13. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

    2015-01-01

    dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

  14. Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans.

    Science.gov (United States)

    Semighini, Camile P; von Zeska Kress Fagundes, Márcia Regina; Ferreira, Joseane Cristina; Pascon, Renata Castiglioni; de Souza Goldman, Maria Helena; Goldman, Gustavo Henrique

    2003-06-01

    The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti-topoisomerase I drug camptothecin. We have used the scaANBS1 cDNA as a bait in a yeast two-hybrid screening and report the identification of the A. nidulans Mre11 homologue (mreA). The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents. Septation in A. nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex. scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra-S-phase checkpoint. ScaANBS1 also participates in G2-M checkpoint control upon DNA damage caused by MMS. In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nidulans. Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.

  15. DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

    2007-12-01

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  16. DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

    2008-02-21

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  17. Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    M. Pádula

    1999-09-01

    Full Text Available In the present study, we analyzed DNA damage induced by phycocyanin (PHY in the presence of visible light (VL using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

  18. Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

    Directory of Open Access Journals (Sweden)

    Silvia Sterpone

    2010-01-01

    Full Text Available It is well known that ionizing radiation (IR can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER. In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.

  19. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Rezaei, Nousin; Liontos, Michalis

    2006-01-01

    Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest......, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks...... and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression....

  20. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  1. Radiation induced DNA damage and repair in mutagenesis

    International Nuclear Information System (INIS)

    Strniste, G.F.; Chen, D.J.; Okinaka, R.T.

    1987-01-01

    The central theme in cellular radiobiological research has been the mechanisms of radiation action and the physiological response of cells to this action. Considerable effort has been directed toward the characterization of radiation-induced DNA damage and the correlation of this damage to cellular genetic change that is expressed as mutation or initiating events leading to cellular transformation and ultimately carcinogenesis. In addition, there has been a significant advancement in their understanding of the role of DNA repair in the process of mutation leading to genetic change in cells. There is extensive literature concerning studies that address radiation action in both procaryotic and eucaryotic systems. This brief report will make no attempt to summarize this voluminous data but will focus on recent results from their laboratory of experiments in which they have examined, at both the cellular and molecular levels, the process of ionizing radiation-induced mutagenesis in cultured human cells

  2. Influence of the complexity of radiation-induced DNA damage on enzyme recognition

    International Nuclear Information System (INIS)

    Palmer, Philip

    2002-01-01

    Ionising radiation is unique in inducing DNA clustered damage together with the simple isolated lesions. Understanding how these complex lesions are recognised and repaired by the cell is key to understanding the health risks associated with radiation exposure. This study focuses on whether ionising radiation-induced complex single-strand breaks (SSB) are recognised by DNA-PK and PARP, and whether the complexity of DSB influence their ligation by either DNA ligase lV/XRCC4 (LX) complex or T4 DNA ligase. Plasmid DNA, irradiated in aqueous solution using sparsely ionising γ-rays and densely ionising α-particles produce different yields of complex DNA damages, used as substrates for in vitro DNA-PK and PARP activity assays. The activity of DNA-PK to phosphorylate a peptide was determined using HF19 cell nuclear extracts as a source of DNA-PK. PARP ADP-ribosylation activity was determined using purified PARP enzyme. The activation of DNA-PK and PARP by irradiated DNA is due to SSB and not the low yield of DSB (linear plasmid DNA <10%). A ∼2 fold increase in DNA-PK activation and a ∼3-fold reduction in PARP activity seen on increasing the ionising density of the radiation (proportion of complex damage) are proposed to reflect changes in the complexity of SSB and may relate to damage signalling. Complex DSB synthesised as double-stranded oligonucleotides, with a 2 bp 5'-overhang, and containing modified lesions, 8-oxoguanine and abasic sites, at known positions relative to the termini were used as substrates for in vitro ligation by DNA ligase IV/XRCC4 or T4 ligase. The presence of a modified lesion 2 or 3 bp but not 4 bp from the 3'-termini and 2 or 6 bp from the 5'-termini caused a drastic reduction in the extent of ligation. Therefore, the presence of modified lesions near to the termini of a DSB may compromise their rejoining by non-homologous end-joining (NHEJ) involving the LX complex. (author)

  3. Proton-induced direct and indirect damage of plasmid DNA

    Czech Academy of Sciences Publication Activity Database

    Vyšín, Luděk; Pachnerová Brabcová, Kateřina; Štěpán, V.; Moretto-Capelle, P.; Bugler, B.; Legube, G.; Cafarelli, P.; Casta, R.; Champeaux, J. P.; Sence, M.; Vlk, M.; Wagner, Richard; Štursa, Jan; Zach, Václav; Incerti, S.; Juha, Libor; Davídková, Marie

    2015-01-01

    Roč. 54, č. 3 (2015), s. 343-352 ISSN 0301-634X R&D Projects: GA ČR GA13-28721S; GA MŠk LD12008; GA MŠk LM2011019 Institutional support: RVO:68378271 ; RVO:61389005 Keywords : proton radiation * DNA plasmid * direct and indirect effects * clustered damage * repair enzymes Subject RIV: BO - Biophysics Impact factor: 1.923, year: 2015

  4. Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

    Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

  5. Nickel exposure induces oxidative damage to mitochondrial DNA in Neuro2a cells: the neuroprotective roles of melatonin.

    Science.gov (United States)

    Xu, Shang-Cheng; He, Min-Di; Lu, Yong-Hui; Li, Li; Zhong, Min; Zhang, Yan-Wen; Wang, Yuan; Yu, Zheng-Ping; Zhou, Zhou

    2011-11-01

    Recent studies suggest that oxidative stress and mitochondrial dysfunction play important roles in the neurotoxicity of nickel. Because mitochondrial DNA (mtDNA) is highly vulnerable to oxidative stress and melatonin can efficiently protect mtDNA against oxidative damage in various pathological conditions, the aims of this study were to determine whether mtDNA oxidative damage was involved in the neurotoxicity of nickel and to assay the neuroprotective effects of melatonin in mtDNA. In this study, we exposed mouse neuroblastoma cell lines (Neuro2a) to different concentrations of nickel chloride (NiCl(2), 0.125, 0.25, and 0.5 mm) for 24 hr. We found that nickel significantly increased reactive oxygen species (ROS) production and mitochondrial superoxide levels. In addition, nickel exposure increased mitochondrial 8-hydroxyguanine (8-OHdG) content and reduced mtDNA content and mtDNA transcript levels. Consistent with this finding, nickel was found to destroy mtDNA nucleoid structure and decrease protein levels of Tfam, a key protein component for nucleoid organization. However, all the oxidative damage to mtDNA induced by nickel was efficiently attenuated by melatonin pretreatment. Our results suggest that oxidative damage to mtDNA may account for the neurotoxicity of nickel. Melatonin has great pharmacological potential in protecting mtDNA against the adverse effects of nickel in the nervous system. © 2011 John Wiley & Sons A/S.

  6. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    Science.gov (United States)

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

  7. Identification of β Clamp-DNA Interaction Regions That Impair the Ability of E. coli to Tolerate Specific Classes of DNA Damage.

    Science.gov (United States)

    Nanfara, Michael T; Babu, Vignesh M P; Ghazy, Mohamed A; Sutton, Mark D

    The E. coli dnaN-encoded β sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of in vitro experiments indicate that the loading of β clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to E. coli viability, as well as the ability of the β clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of β clamp-DNA interactions to the ability of E. coli to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct β clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the β clamp through which the DNA template threads. Twenty of these 22 dnaN mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting hda function. Taken together, these results demonstrate that discrete β clamp-DNA interaction regions contribute to the ability of E. coli to tolerate specific classes of DNA damage.

  8. Factors that influence telomeric oxidative base damage and repair by DNA glycosylase OGG1

    DEFF Research Database (Denmark)

    Rhee, David B; Ghosh, Avik; Lu, Jian

    2011-01-01

    Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major...... contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors...... affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non...

  9. An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

    DEFF Research Database (Denmark)

    Johansson, Clara; Møller, Peter; Forchhammer, Lykke

    2010-01-01

    The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to dif...

  10. rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint

    NARCIS (Netherlands)

    Walworth, N.C.; Bernards, R.A.

    1996-01-01

    Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56chk1. DNA damage induced by ultraviolet

  11. PIG3 Functions in DNA Damage Response through Regulating DNA-PKcs Homeostasis

    OpenAIRE

    Li, Bing; Shang, Zeng-Fu; Yin, Jiao-Jiao; Xu, Qin-Zhi; Liu, Xiao-Dan; Wang, Yu; Zhang, Shi-Meng; Guan, Hua; Zhou, Ping-Kun

    2013-01-01

    The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnor...

  12. Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage.

    Science.gov (United States)

    Ding, Ning; Bonham, Emily M; Hannon, Brooke E; Amick, Thomas R; Baylin, Stephen B; O'Hagan, Heather M

    2016-06-01

    At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein-protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  13. Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

    International Nuclear Information System (INIS)

    Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

    2011-01-01

    The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

  14. Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

    Energy Technology Data Exchange (ETDEWEB)

    Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita [Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu (Estonia); Kivisaar, Maia, E-mail: maiak@ebc.ee [Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu (Estonia)

    2011-09-01

    The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif{sup r} mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    International Nuclear Information System (INIS)

    Roper, Katherine; Coverley, Dawn

    2012-01-01

    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 naïve 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 naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve 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: ► A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. ► Damage-activated extracts impose the cellular response to DNA damage on naïve nuclei. ► PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. ► Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. ► LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

  17. Oxidative DNA damage caused by pulsed discharge with cavitation on the bactericidal function

    Science.gov (United States)

    Kudo, Ken-ichi; Ito, Hironori; Ihara, Satoshi; Terato, Hiroaki

    2015-09-01

    Plasma-based techniques are expected to have practical use for wastewater purification with a potential for killing contaminated microorganisms and degrading recalcitrant materials. In the present study, we analysed oxidative DNA damage in bacterial cells treated by the plasma to unveil its mechanisms in the bactericidal process. Escherichia coli cell suspension was exposed to the plasma induced by applying an alternating-current voltage of about 1 kV with bubbling formed by water-cavitation, termed pulsed discharge with cavitation. Chromosomal DNA damage, such as double strand break (DSB) and oxidative base lesions, increased proportionally with the applied energy, as determined by electrophoretic and mass spectrometric analyses. Among the base lesions identified, the yields of 8-hydroxyguanine (8-OH-G) and 5-hydroxycytosine (5-OH-C) in chromosomal DNA increased by up to 4- and 15-fold, respectively, compared to untreated samples. The progeny DNA sequences, derived from plasmid DNA exposed to the plasma, indicated that the production rate of 5-OH-C exceeded that of 8-OH-G, as G:C to A:T transitions accounted for 65% of all base changes, but only a few G:C to T:A transversions were observed. The cell viabilities of E. coli cells decreased in direct proportion to increases in the applied energy. Therefore, the plasma-induced bactericidal mechanism appears to relate to oxidative damage caused to bacterial DNA. These results were confirmed by observing the generation of hydroxyl radicals and hydrogen peroxide molecules following the plasma exposure. We also compared our results with the plasma to those obtained with 137Cs γ-rays, as a well-known ROS generator to confirm the DNA-damaging mechanism involved.

  18. Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast.

    Science.gov (United States)

    Herbert, Sébastien; Brion, Alice; Arbona, Jean-Michel; Lelek, Mickaël; Veillet, Adeline; Lelandais, Benoît; Parmar, Jyotsana; Fernández, Fabiola García; Almayrac, Etienne; Khalil, Yasmine; Birgy, Eleonore; Fabre, Emmanuelle; Zimmer, Christophe

    2017-09-01

    DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility. © 2017 The Authors.

  19. Recognition of DNA damage and induction of repair proteins in deinococcus radiodurans

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Katsuya; Kikuchi, Masahiro; Narumi, Issay [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2002-03-01

    In Escherichia coli, the inducible DNA repair system (the SOS system) is regulated by two key proteins; RecA and LexA. Although SOS-like processes have been documented in a wide variety of eubacterial species. The involvement of RecA and LexA in the SOS response is poorly understood in Deinococcus radiodurans. As expression of the deinococcal recA is enhanced after irradiation, the recA seems to be a member of a DNA damage response regulon in D. radiodurans. By investigating the function of deinococcal proteins, we found that LexA is not involved in the regulation of RecA in D. radiodurans. This led us to speculate that D. radiodurans has an alternative DNA damage response mechanism with which to control recA expression. We discovered that a novel protein regulates the expression of recA. The novel regulatory protein (designated PprI) also controls the induction of pprA following irradiation. We also found that the co-protease activity rather than recombination activity of RecA contributes to the radiation resistance in D. radiodurans. The D. radiodurans genome encodes a second but diverged copy of LexA (designated LexA2). Analysis of LexA2 indicated that still-unknown repair gene are down-regulated by LexA2. Thus, D. radiodurans possesses unique mechanisms of DNA damage recognition and repair gene induction. (author)

  20. The impact of impaired DNA damage responses on cells, tissues and organisms

    NARCIS (Netherlands)

    Yi, Xia

    2007-01-01

    Current cancer therapies rely mainly on DNA damaging insults (irradiation, DNA alkylating agents, DNA synthesis inhibitors etc.). The rationale behind these treatments is that rapidly growing cancer cells suffer more from DNA damaging insults. Unfortunately, the majority of current therapies fail to

  1. Mitochondrial DNA Damage and Animal Longevity: Insights from Comparative Studies

    Directory of Open Access Journals (Sweden)

    Reinald Pamplona

    2011-01-01

    Full Text Available Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA. ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

  2. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

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

    Science.gov (United States)

    Hamperl, Stephan; Cimprich, Karlene A

    2014-07-01

    Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Radiation track, DNA damage and response—a review

    Science.gov (United States)

    Nikjoo, H.; Emfietzoglou, D.; Liamsuwan, T.; Taleei, R.; Liljequist, D.; Uehara, S.

    2016-11-01

    The purpose of this paper has been to review the current status and progress of the field of radiation biophysics, and draw attention to the fact that physics, in general, and radiation physics in particular, with the aid of mathematical modeling, can help elucidate biological mechanisms and cancer therapies. We hypothesize that concepts of condensed-matter physics along with the new genomic knowledge and technologies and mechanistic mathematical modeling in conjunction with advances in experimental DNA (Deoxyrinonucleic acid molecule) repair and cell signaling have now provided us with unprecedented opportunities in radiation biophysics to address problems in targeted cancer therapy, and genetic risk estimation in humans. Obviously, one is not dealing with ‘low-hanging fruit’, but it will be a major scientific achievement if it becomes possible to state, in another decade or so, that we can link mechanistically the stages between the initial radiation-induced DNA damage; in particular, at doses of radiation less than 2 Gy and with structural changes in genomic DNA as a precursor to cell inactivation and/or mutations leading to genetic diseases. The paper presents recent development in the physics of radiation track structure contained in the computer code system KURBUC, in particular for low-energy electrons in the condensed phase of water for which we provide a comprehensive discussion of the dielectric response function approach. The state-of-the-art in the simulation of proton and carbon ion tracks in the Bragg peak region is also presented. The paper presents a critical discussion of the models used for elastic scattering, and the validity of the trajectory approach in low-electron transport. Brief discussions of mechanistic and quantitative aspects of microdosimetry, DNA damage and DNA repair are also included as developed by the authors’ work.

  5. Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans

    DEFF Research Database (Denmark)

    Møller, P; Loft, S; Lundby, C

    2001-01-01

    The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test...... oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity...

  6. Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA.

    Science.gov (United States)

    Adam, Salomé; Polo, Sophie E; Almouzni, Geneviève

    2013-09-26

    Understanding how to recover fully functional and transcriptionally active chromatin when its integrity has been challenged by genotoxic stress is a critical issue. Here, by investigating how chromatin dynamics regulate transcriptional activity in response to DNA damage in human cells, we identify a pathway involving the histone chaperone histone regulator A (HIRA) to promote transcription restart after UVC damage. Our mechanistic studies reveal that HIRA accumulates at sites of UVC irradiation upon detection of DNA damage prior to repair and deposits newly synthesized H3.3 histones. This local action of HIRA depends on ubiquitylation events associated with damage recognition. Furthermore, we demonstrate that the early and transient function of HIRA in response to DNA damage primes chromatin for later reactivation of transcription. We propose that HIRA-dependent histone deposition serves as a chromatin bookmarking system to facilitate transcription recovery after genotoxic stress. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Monitoring of DNA and cytogenetic damage in lymphocytes from persons with skin cancer diseases

    International Nuclear Information System (INIS)

    Cebulska-Wasilewska, A.; Dyga, W.; Krasnowolski, S.; Wierzewska, A.; Budzanowska, E.

    1999-01-01

    There is a lot of interest in the studies that would help to understand whether there is a casual association between cancer and various types of molecular or cytogenetic damage detected in human cells. One major oncogenesis process is activation of proto-oncogenes by point mutations or chromosomal translocation. There are substantial evidence that indicates that the loss of heterozygosity of certain chromosomes is involved in human cancerogenesis. Our study aimed to elicit the possible association between cancer and DNA and cytogenetic abnormalities induced in lymphocytes of people bearing various categories of skin cancer cells. Fresh blood was collected by venipuncture from 25 individuals (including nine prior to cancer treatment). All patients were nonsmoking males, however 42.3 % of them were former smokers. Blood samples were divided into two parts and in the first part of samples cytogenetic studies were performed immediately, while from the second part lymphocytes were isolated and stored at -70 o C for further studies in vitro. In the later one a single cell gel electrophoresis assay (SCGE) known as a Comet assay was performed to study individual susceptibility to the induction of DNA damage by UV or radiation and to estimate variability in cellular repair capabilities. An average of 220 per sample of good metaphase spreads in the first mitotic division, and 100 per sample in the second division, were accepted for analysis of cytogenetic damage. Chromosome and chromatid type aberrations were scored in the cells in the first mitosis and expressed as total aberration frequency including gaps and excluding gaps. Sister chromatid exchanges, high frequency cells and proliferative rate index were screened and evaluated in the second mitosis. Each of the patient revealed exceeding in at least one of the cytogenetic biomarkers level from the biomarker's level detected in a reference group. In order to estimate susceptibility of people to environmentally induced

  8. Pro-oxidant induced DNA damage in human lymphoblastoid cells: homeostatic mechanisms of genotoxic tolerance.

    Science.gov (United States)

    Seager, Anna L; Shah, Ume-Kulsoom; Mikhail, Jane M; Nelson, Bryant C; Marquis, Bryce J; Doak, Shareen H; Johnson, George E; Griffiths, Sioned M; Carmichael, Paul L; Scott, Sharon J; Scott, Andrew D; Jenkins, Gareth J S

    2012-08-01

    Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H(2)O(2)), potassium bromate (KBrO(3)), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H(2)O(2) and KBrO(3), but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair-focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H(2)O(2)-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H(2)O(2) dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in "genotoxic tolerance."

  9. Maintaining Genome Stability in Defiance of Mitotic DNA Damage

    Science.gov (United States)

    Ferrari, Stefano; Gentili, Christian

    2016-01-01

    The implementation of decisions affecting cell viability and proliferation is based on prompt detection of the issue to be addressed, formulation and transmission of a correct set of instructions and fidelity in the execution of orders. While the first and the last are purely mechanical processes relying on the faithful functioning of single proteins or macromolecular complexes (sensors and effectors), information is the real cue, with signal amplitude, duration, and frequency ultimately determining the type of response. The cellular response to DNA damage is no exception to the rule. In this review article we focus on DNA damage responses in G2 and Mitosis. First, we set the stage describing mitosis and the machineries in charge of assembling the apparatus responsible for chromosome alignment and segregation as well as the inputs that control its function (checkpoints). Next, we examine the type of issues that a cell approaching mitosis might face, presenting the impact of post-translational modifications (PTMs) on the correct and timely functioning of pathways correcting errors or damage before chromosome segregation. We conclude this essay with a perspective on the current status of mitotic signaling pathway inhibitors and their potential use in cancer therapy. PMID:27493659

  10. Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Borrego-Soto, Gissela; Ortiz-Lopez, Rocio; Rojas-Martinez, Augusto, E-mail: arojasmtz@gmail.com, E-mail: augusto.rojasm@uanl.mx [Departamento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León (Mexico)

    2015-10-15

    Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity. (author)

  11. Regulated Proteolysis of DNA Polymerase Eta During the DNA Damage Response in C. elegans

    Science.gov (United States)

    Kim, Seung-Hwan; Michael, W. Matthew

    2009-01-01

    SUMMARY Both the POLH-1 (pol eta) trans-lesion synthesis DNA polymerase and the GEI-17 SUMO E3 ligase are essential for the efficient replication of damaged chromosomes in C. elegans embryos. Here, we study how POLH-1 is regulated during a DNA damage response in these embryos. We report that DNA damage triggers the degradation of POLH-1, and that degradation is mediated by the Cul4-Ddb1-Cdt2 (CRL4-Cdt2) pathway that has previously been shown to degrade the replication factor Cdt1 during S phase. We also show that GEI-17 protects POLH-1 from CRL4-Cdt2 mediated destruction, until after it has performed its function in TLS, and this is likely via SUMOylation of POLH-1. These studies reveal that POLH-1 undergoes DNA damage-induced proteolysis, and that GEI-17 regulates the timing of this proteolysis. Implications for how this system may control the removal of POLH-1 from replication forks after TLS are discussed. PMID:19111656

  12. Regulated proteolysis of DNA polymerase eta during the DNA-damage response in C. elegans.

    Science.gov (United States)

    Kim, Seung-Hwan; Michael, W Matthew

    2008-12-26

    Both the POLH-1 (pol eta) translesion synthesis (TLS) DNA polymerase and the GEI-17 SUMO E3 ligase are essential for the efficient replication of damaged chromosomes in Caenorhabditis elegans embryos. Here we study how POLH-1 is regulated during a DNA-damage response in these embryos. We report that DNA damage triggers the degradation of POLH-1 and that degradation is mediated by the Cul4-Ddb1-Cdt2 (CRL4-Cdt2) pathway that has previously been shown to degrade the replication factor Cdt1 during S phase. We also show that GEI-17 protects POLH-1 from CRL4-Cdt2-mediated destruction until after it has performed its function in TLS, and this is likely via SUMOylation of POLH-1. These studies reveal that POLH-1 undergoes DNA-damage-induced proteolysis and that GEI-17 regulates the timing of this proteolysis. Implications for how this system may control the removal of POLH-1 from replication forks after TLS are discussed.

  13. Influence of the OGG1 Ser326Cys polymorphism on oxidatively damaged DNA and repair activity

    DEFF Research Database (Denmark)

    Jensen, Annie; Løhr, Mille; Eriksen, Louise

    2012-01-01

    Oxidatively damaged DNA base lesions are considered to be mainly repaired by 8-oxoguanine DNA glycosylase (OGG1) mediated pathways. We investigated the effect of the OGG1 Ser326Cys polymorphism on the level and repair of oxidatively damaged DNA in mononuclear blood cells (MNBC) by means of the co......Oxidatively damaged DNA base lesions are considered to be mainly repaired by 8-oxoguanine DNA glycosylase (OGG1) mediated pathways. We investigated the effect of the OGG1 Ser326Cys polymorphism on the level and repair of oxidatively damaged DNA in mononuclear blood cells (MNBC) by means...

  14. Decreased drug accumulation and increased tolerance to DNA damage in tumor cells with a low level of cisplatin resistance.

    Science.gov (United States)

    Lanzi, C; Perego, P; Supino, R; Romanelli, S; Pensa, T; Carenini, N; Viano, I; Colangelo, D; Leone, R; Apostoli, P; Cassinelli, G; Gambetta, R A; Zunino, F

    1998-04-15

    In an attempt to examine the cellular changes associated with cisplatin resistance, we selected a cisplatin-resistant (A43 1/Pt) human cervix squamous cell carcinoma cell line following continuous in vitro drug exposure. The resistant subline was characterized by a 2.5-fold degree of resistance. In particular, we investigated the expression of cellular defence systems and other cellular factors probably involved in dealing with cisplatin-induced DNA damage. Resistant cells exhibited decreased platinum accumulation and reduced levels of DNA-bound platinum and interstrand cross-link frequency after short-term drug exposure. Analysis of the effect of cisplatin on cell cycle progression revealed a cisplatin-induced G2M arrest in sensitive and resistant cells. Interestingly, a slowdown in S-phase transit was found in A431/Pt cells. A comparison of the ability of sensitive and resistant cells to repair drug-induced DNA damage suggested that resistant cells were able to tolerate higher levels of cisplatin-induced DNA damage than their parental counterparts. Analysis of the expression of proteins involved in DNA mismatch repair showed a decreased level of MSH2 in resistant cells. Since MSH2 seems to be involved in recognition of drug-induced DNA damage, this change may account for the increased tolerance to DNA damage observed in the resistant subline. In conclusion, the involvement of accumulation defects and the increased tolerance to cisplatin-induced DNA damage in these cisplatin-resistant cells support the notion that multiple changes contribute to confer a low level of cisplatin resistance.

  15. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  16. DNA damage in Human Limbal Epithelial Cells expanded ex vivo.

    Directory of Open Access Journals (Sweden)

    Yolanda Lorenzo Corrales

    2015-04-01

    Full Text Available Limbal stem cell deficiency, secondary to insults and diseases, may be treated by transplantation of ex vivo engineered epithelial grafts. We here present preliminary data on levels of cellular DNA damage in grafts produced in two different types of culture medium. Cultures were initiated using corneo-limbal donor tissue after removal of the central area for transplant purposes. Explants (approx. 2x2 mm were positioned epithelial side down on tissue culture treated polyester membranes and expanded for four weeks in Dulbecco’s Modified Eagle Medium F12 Nutrient Mixture (Ham [DMEM/F12 (1:1] with either (1 H. medium; 10% human serum or (2 COM; 5% fetal bovine serum (FBS, Epidermal Growth Factor (EGF, insulin-transferrin-sodiumselenzine (ITS , cholera toxin-A, dimethyl sulfoxide (DMSO and hydrocortisone. Cells were dissociated using Trypsin-EDTA (0.05% for 30 min., the enzyme activity was inhibited by medium and serum. The cell suspension was transferred to tubes on ice and processed using the Comet Assay. Duplicate samples from each culture were analyzed in each assay by visual scoring. Using a fluorescence microscope, 100 comets (50 from each gel were classified into five categories, 0-4, representing increasing relative tail intensities. Summing the scores (0-4 of 100 comets therefore gives an overall score of between 0 and 400 arbitrary units. Preliminary data show some levels of DNA damage in cells dissociated from the grafts regardless of the type of culture medium used. Anyway more experiments with other donors have to be done to have some conclusions. Recent studies have shown that medium with human serum equally support production of grafts containing differentiated as well as undifferentiated cells suitable for clinical transplantation. Preliminary data from our experiments indicate that levels of molecular damage to the DNA do not increase in cells cultured in H. medium despite its lacks of complexity.

  17. Chromatin structure influence the sensitivity of DNA to ionizing radiation induced DNA damage

    International Nuclear Information System (INIS)

    Gupta, Sanjay

    2016-01-01

    Chromatin acts as a natural hindrance in DNA-damage recognition, repair and recovery. Histone and their variants undergo differential post-translational modification(s) and regulate chromatin structure to facilitate DNA damage response (DDR). During the presentation we will discuss the importance of chromatin organization and histone modification(s) during IR-induced DNA damage response in human liver cells. Our data shows G1-phase specific decrease of H3 serine10 phosphorylation in response to DNA damage is coupled with chromatin compaction in repair phase of DDR. The loss of H3Ser10P during DNA damage shows an inverse correlation with gain of γH2AX from a same mono-nucleosome in a dose-dependent manner. The loss of H3Ser10P is a universal phenomenon as it is independent of origin of cell lines and nature of genotoxic agents in G1 phase cells. The reversible reduction of H3Ser10P is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. The present study suggests distinct reversible histone marks are associated with G1-phase of cell cycle and plays a critical role in chromatin organization which may facilitate differential sensitivity against radiation. Thus, the study raises the possibility of combinatorial modulation of H3Ser10P and histone acetylation with specific inhibitors to target the radio-resistant cancer cells in G1-phase and thus may serve as promising targets for cancer therapy. (author)

  18. Investigation of the reactions of histone protein hydroperoxides and their role in DNA damage

    International Nuclear Information System (INIS)

    Luxford, C.; Dean, R.T.; Davies, M.J.

    1998-01-01

    Free radical attack on DNA results in base changes, cross-linking and strand cleavage leading to mutations if unrepaired. Histone proteins are intimately involved in DNA packaging and are excellent candidates for investigating DNA damage arising from protein-OOH-derived radicals. This study aimed (i) to investigate the formation of hydroperoxide on the linker histone H1 via radical reactions in the presence of O 2 ; (ii) to examine the radicals formed from transition metal ion-catalyzed breakdown of histone H1-OOH and (iii) to determine whether histone H1-OOH-derived radicals can damage DNA and free bases. (i) Histone H1 solutions were γ-irradiated ( 60 Co source) in the presence of O 2 and histone H1-OOH concentrations determined using a manual iodometric assay. Formation ( histone H1-OOH was dose-dependent and, in the absence of light or transition metal ions these hydroperoxides were found to be very stable (half life of 24 hours at 4degC ). (ii) Electron Paramagnetic Resonance (EPR) spectroscopy and spin trapping was used t investigate the Cu + -catalyzed breakdown of histone H1-OOH to form histone H1 protein side chain and -backbone carbon-centred radicals. Further EPR/spin trapping experiments showed that histone H1-OOH-derived radicals can oxidise pyrimidine bases (eg. uridine with the resultant trapping of three radical species; two pyrimidine radicals, C5-yl and Ct yl adducts (via addition of histone H1-OOH-derived radicals to the C5-C6 double bond o the pyrimidine ring) and an acyl radical adduct, whose origin is currently unknown. (iii) Damage to DNA and 2'-deoxyguanosine after reaction of histone H1-OOH-derive radicals were detected and quantified using HPLC (with EC and UV detection). We have identified 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as a significant product ( histone H1-OOH-derived oxidative DNA modification. Increasing histone H1-OOH concentrations resulted in a concomitant increase in the amount of 8-oxodG formed. Our studies show

  19. Personal exposure to ultrafine particles and oxidative DNA damage

    DEFF Research Database (Denmark)

    Vinzents, Peter S; Møller, Peter; Sørensen, Mette

    2005-01-01

    10), nitrous oxide, nitrogen dioxide, carbon monoxide, and/or number concentration of UFPs at urban background or busy street monitoring stations was not a significant predictor of DNA damage, although personal UFP exposure was correlated with urban background concentrations of CO and NO2...... the morning after exposure measurement. Cumulated outdoor and cumulated indoor exposures to UFPs each were independent significant predictors of the level of purine oxidation in DNA but not of strand breaks. Ambient air concentrations of particulate matter with an aerodynamic diameter of ..., particularly during bicycling in traffic. The results indicate that biologic effects of UFPs occur at modest exposure, such as that occurring in traffic, which supports the relationship of UFPs and the adverse health effects of air pollution....

  20. Online imaging of initial DNA damages at the PTB microbeam

    International Nuclear Information System (INIS)

    Giesen, U.; Langner, F.; Mielke, C.; Mosconi, M.; Dirks, W. G.

    2011-01-01

    In an inter-disciplinary collaboration of Physikalisch-Technische Bundesanstalt (PTB), German Collection of Microorganisms and Cell Cultures (DSMZ) and Heinrich-Heine Univ., live-cell imaging has been established at the charged-particle microbeam facility of PTB. Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MRE11, MSH2, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterise and use these highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions. (authors)

  1. 8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage

    DEFF Research Database (Denmark)

    Loft, S; Fischer-Nielsen, A; Jeding, I B

    1993-01-01

    and in various laboratory animals, including dog, pig, and rat. Previously, other groups have used comparable HPLC methods or gas chromatography-mass spectrometry with selective ion monitoring for measuring the excretion of 8OHdG in humans, rats, mice, and monkeys. In the 169 humans studied so far, the average 8......-hydroxydeoxyguanosine (8OHdG) has been proposed as a noninvasive biomarker of oxidative DNA damage in humans in vivo. We have developed a three-dimensional HPLC analysis with electrochemical detection for the analysis of 8OHdG in urine and studied factors affecting the excretion of this biomarker in 83 healthy humans...

  2. Are all phytochemicals useful in the preventing of DNA damage?

    Science.gov (United States)

    Bacanlı, Merve; Aydın, Sevtap; Başaran, A Ahmet; Başaran, Nurşen

    2017-11-01

    Phytochemicals derived from natural plants have been used commonly for the prevention and/or treatment of different diseases due to the belief of their safety. Many plant species synthesize toxic chemicals. New natural chemicals are being discovered but their toxic effects are unknown. Phytochemicals have been regarded as possible antioxidants. But on the other hand it is suggested that various phenolic antioxidants can display pro-oxidant properties at high doses. In this review, the role of some phytochemicals (epigallocathecin gallate, carvacrol, galangin, limonene, lycopene, naringin, puerarin, terpinene, thymol and ursolic acid) on the prevention of DNA damage will be discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Lymphocyte DNA damage in elevator manufacturing workers in Guangzhou, China.

    Science.gov (United States)

    Lam, Tai Hing; Zhu, Chang Qi; Jiang, Chao Qiang

    2002-03-25

    To study the effect of smoking, passive smoking, alcohol drinking, and occupational exposure to low level of benzene on DNA strand breaks in elevator manufacturing workers in Guangzhou, China. Three hundred and fifty-nine workers (252 men and 107 women) of a modern elevator manufacturing factory, 205 were from production departments and 154 from managerial department. Information on the workers' health conditions, smoking, passive smoking, alcohol consumption and occupational exposure history was collected by personal interview. Lymphocyte DNA damage was measured by the Comet assay. None of the women smoked and 20.6% of the men were daily smokers. In non-smokers, the prevalence of passive smoking at work was 25% for men and 11.2% for women, and at home, 37.8 and 48.6%, respectively. Smoking significantly increased tail moment (P<0.001). Daily smokers had the largest tail moment (geometric mean, 95% CI) (0.93 microm (0.81-0.94)), followed by occasional smokers (0.76 microm (0.59-0.95)), ex-smokers (0.70 microm (0.58-0.85)), and never smokers (0.56 microm (0.53-0.60)). Tail moment increased significantly with daily tobacco consumption (cigarettes per day) (r=0.26, P<0.001) after adjusting for age, gender, occupational exposure, passive smoking, and drinking. Analysis of covariance (ANCOVA) showed that smoking (P<0.001), passive smoking at home (P=0.026), occupational exposure (P<0.001), male gender (P<0.001), and age (P=0.001) had independent effects on tail moment, whereas passive smoking at work and alcohol drinking had no significant effect. Smoking, passive smoking at home, male gender, age and occupational exposure independently increased lymphocyte DNA strand breaks. The presence of excess DNA damage under low level of occupational exposure to benzene or other solvents suggest that the current allowance concentrations may not be safe to prevent genotoxicity.

  4. Monitoring of DNA and cytogenetic damage in lymphocytes in patients with skin cancer disease

    International Nuclear Information System (INIS)

    Cebulska-Wasilewska, A.; Dyga, W.; Krasnowolski, S.; Wierzewska, A.; Budzanowska, E.

    1999-01-01

    One major oncogenesis process is activation of proto-oncogenes by point mutations or chromosomal translocations. There is substantial evidence that indicates that human carcinogenesis involves loss of heterozygosity of certain chromosomes. Our study aimed at searching the possible association between cancer and DNA and cytogenetic abnormalities induced in lymphocytes of people with various categories of skin cancer cells. Fresh blood was collected by venepuncture from 25 individuals (including nine prior to cancer treatment). All patients were nonsmoking males, however 42.3% of them were former smokers. Blood samples were divided into two parts; in the first part of samples cytogenetic studies were performed immediately, while lymphocytes from the other part were isolated and stored at -70 0C for further studies in vitro. A single cell gel electrophoresis assay (SCGE), known as a Comet assay, was performed on them to study individual susceptibility to the induction of DNA damage by UV or radiation and to estimate variability in cellular repair capabilities. On average 220 good metaphase spreads per sample in the first mitotic division, and 100 spreads per sample in the second division were accepted for analysis of the cytogenetic damage. Chromosome and chromatid type aberrations were scored in the cells in the first mitosis, and expressed as total aberration frequency including and excluding gaps. Sister chromatid exchanges , high frequency cells and proliferating rate index were screened and evaluated in the second mitosis. Each patient showed a level exceeding (in at least one of the cytogenetic biomarker) the biomarker level in a reference group. In order to estimate susceptibility of people to environmentally induced damage, the isolated lymphocytes were irradiated with 2 Gy dose of X-rays or 6 J/m 2 of UV radiation, and the single cell gel electrophoresis (SCGE assay) was performed. To compare various individual capabilities to repair the induced damage

  5. Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System*

    Science.gov (United States)

    Lindsey-Boltz, Laura A.; Kemp, Michael G.; Reardon, Joyce T.; DeRocco, Vanessa; Iyer, Ravi R.; Modrich, Paul; Sancar, Aziz

    2014-01-01

    DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response. PMID:24403078

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

    OpenAIRE

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

    2014-01-01

    The DNA damage response (DDR) maintains genomic integrity following DNA damage to prevent cancer and developmental disorders. The DDR operates in part through controlling localization of factors to chromatin. Here, we detail an interaction between the DDR protein NBS1 and TCOF1, a nucleolar protein mutated in Treacher Collins syndrome that regulates ribosomal DNA transcription. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and independent on the NBS...

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

    Science.gov (United States)

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

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Dietary flavonoid derivatives enhance chemotherapeutic effect by inhibiting the DNA damage response pathway

    International Nuclear Information System (INIS)

    Kuo, Ching-Ying; Zupkó, István; Chang, Fang-Rong; Hunyadi, Attila; Wu, Chin-Chung; Weng, Teng-Song; Wang, Hui-Chun

    2016-01-01

    Flavonoids are the most common group of polyphenolic compounds and abundant in dietary fruits and vegetables. Diet high in vegetables or dietary flavonoid supplements is associated with reduced mortality rate for patients with breast cancer. Many studies have been proposed for mechanisms linking flavonoids to improving chemotherapy efficacy in many types of cancers, but data on this issue is still limited. Herein, we report on a new mechanism through which dietary flavonoids inhibit DNA damage checkpoints and repair pathways. We found that dietary flavonoids could inhibit Chk1 phosphorylation and decrease clonogenic cell growth once breast cancer cells receive ultraviolet irradiation, cisplatin, or etoposide treatment. Since the ATR-Chk1 pathway mainly involves response to DNA replication stress, we propose that flavonoid derivatives reduce the side effect of chemotherapy by improving the sensitivity of cycling cells. Therefore, we propose that increasing intake of common dietary flavonoids is beneficial to breast cancer patients who are receiving DNA-damaging chemotherapy, such as cisplatin or etoposide-based therapy. - Highlights: • First report on inhibition of both DNA damage and repair by dietary flavonoids • Dietary flavonoids inhibit cisplatin- and UV-induced Chk1 phosphorylation. • Flavonoids combined with cisplatin or UV treatment show notable growth inhibition. • Promising treatment proposal for patients who are receiving adjuvant chemotherapy

  9. Impact of paper filtered coffee on oxidative DNA-damage: results of a clinical trial.

    Science.gov (United States)

    Mišík, Miroslav; Hoelzl, Christine; Wagner, Karl-Heinz; Cavin, Christophe; Moser, Beate; Kundi, Michael; Simic, Tanja; Elbling, Leonilla; Kager, Nina; Ferk, Franziska; Ehrlich, Veronika; Nersesyan, Armen; Dušinská, Maria; Schilter, Benoît; Knasmüller, Siegfried

    2010-10-13

    Coffee is among the most frequently consumed beverages worldwide and epidemiological studies indicate that its consumption is inversely related to the incidence of diseases in which reactive oxygen species (ROS) are involved (liver cirrhosis, certain forms of cancer and neurodegenerative disorders). It has been postulated that antioxidant properties of coffee may account for this phenomenon. To find out if consumption of paper filtered coffee which is the most widely consumed form in Central Europe and the US protects humans against oxidative DNA-damage, a controlled intervention trial with a cross-over design was conducted in which the participants (n=38) consumed 800ml coffee or water daily over 5 days. DNA-damage was measured in peripheral lymphocytes in single cell gel electrophoresis assays. The extent of DNA-migration attributable to formation of oxidised purines (formamidopyrimidine glycosylase sensitive sites) was decreased after coffee intake by 12.3% (p=0.006). Biochemical parameters of the redox status (malondialdehyde, 3-nitrotyrosine and the total antioxidant levels in plasma, glutathione concentrations in blood, intracellular ROS levels and the activities of superoxide dismutase and glutathione peroxidase in lymphocytes) were not markedly altered at the end of the trial, also the urinary 8-isoprostaglandine F2α concentrations were not affected. Overall, the results indicate that coffee consumption prevents endogenous formation of oxidative DNA-damage in human, this observation may be causally related to beneficial health effects of coffee seen in earlier studies. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Designing a Single-Molecule Biophysics Tool for Characterising DNA Damage for Techniques that Kill Infectious Pathogens Through DNA Damage Effects.

    Science.gov (United States)

    Miller, Helen; Wollman, Adam J M; Leake, Mark C

    2016-01-01

    Antibiotics such as the quinolones and fluoroquinolones kill bacterial pathogens ultimately through DNA damage. They target the essential type IIA topoisomerases in bacteria by stabilising the normally transient double-strand break state which is created to modify the supercoiling state of the DNA. Here we discuss the development of these antibiotics and their method of action. Existing methods for DNA damage visualisation, such as the comet assay and immunofluorescence imaging can often only be analysed qualitatively and this analysis is subjective. We describe a putative single-molecule fluorescence technique for quantifying DNA damage via the total fluorescence intensity of a DNA origami tile fully saturated with an intercalating dye, along with the optical requirements for how to implement these into a light microscopy imaging system capable of single-molecule millisecond timescale imaging. This system promises significant improvements in reproducibility of the quantification of DNA damage over traditional techniques.

  11. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  12. DNA Damage by Radiation in Tradescantia Leaf Cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-04-15

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  13. Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway

    Science.gov (United States)

    Liang, Dun; Burkhart, Sarah Lyn; Singh, Rakesh Kumar; Kabbaj, Marie-Helene Miquel; Gunjan, Akash

    2012-01-01

    In eukaryotes, multiple genes encode histone proteins that package genomic deoxyribonucleic acid (DNA) and regulate its accessibility. Because of their positive charge, ‘free’ (non-chromatin associated) histones can bind non-specifically to the negatively charged DNA and affect its metabolism, including DNA repair. We have investigated the effect of altering histone dosage on DNA repair in budding yeast. An increase in histone gene dosage resulted in enhanced DNA damage sensitivity, whereas deletion of a H3–H4 gene pair resulted in reduced levels of free H3 and H4 concomitant with resistance to DNA damaging agents, even in mutants defective in the DNA damage checkpoint. Studies involving the repair of a HO endonuclease-mediated DNA double-strand break (DSB) at the MAT locus show enhanced repair efficiency by the homologous recombination (HR) pathway on a reduction in histone dosage. Cells with reduced histone dosage experience greater histone loss around a DSB, whereas the recruitment of HR factors is concomitantly enhanced. Further, free histones compete with the HR machinery for binding to DNA and associate with certain HR factors, potentially interfering with HR-mediated repair. Our findings may have important implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes. PMID:22850743

  14. The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage.

    Science.gov (United States)

    Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A; Gwerder, Myriam; Gutsche, Katrin; Altmeyer, Matthias; Jungmichel, Stephanie; Toledo, Luis I; Fink, Daniel; Rask, Maj-Britt; Grøfte, Merete; Lukas, Claudia; Nielsen, Michael L; Smerdon, Stephen J; Lukas, Jiri; Stucki, Manuel

    2014-08-01

    Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.

  15. Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Dimitra T Stefanou

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

  16. DNA damage response and prostate cancer: defects, regulation and therapeutic implications

    Science.gov (United States)

    Karanika, Styliani; Karantanos, Theodoros; Li, Likun; Corn, Paul G.; Thompson, Timothy C.

    2014-01-01

    DNA damage response (DDR) includes the activation of numerous cellular activities that prevent duplication of DNA lesions and maintain genomic integrity, which is critical for the survival of normal and cancer cells. Specific genes involved in the DDR such as BRCA1/2 and P53 are mutated during prostate cancer progression, while various oncogenic signaling such as Akt and c-Myc are activated, enhancing the replication stress and increasing the genomic instability of cancer cells. These events may render prostate cancer cells particularly sensitive to inhibition of specific DDR pathways, such as PARP in homologous recombination (HR) DNA repair and Chk1 in cell cycle checkpoint and DNA repair, creating opportunities for synthetic lethality or synergistic cytotoxicity. Recent reports highlight the critical role of androgen receptor (AR) as a regulator of DDR genes, providing a rationale for combining DNA-damaging agents or targeted DDR inhibitors with hormonal manipulation or AR inhibition as treatment for aggressive disease. The aims of this review are to discuss specific DDR defects in prostate cancer that occur during disease progression, to summarize recent advances in understanding the regulation of DDR in prostate cancer, and to present potential therapeutic opportunities through combinational targeting of the intact components of DDR signaling pathways. PMID:25132269

  17. DNA damage and cell cycle events implicate cerebellar dentate nucleus neurons as targets of Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Yang Yan

    2010-12-01

    Full Text Available Abstract Background Although the cerebellum is considered to be predominantly involved in fine motor control, emerging evidence documents its participation in language, impulsive behavior and higher cognitive functions. While the specific connections of the cerebellar deep nuclei (CDN that are responsible for these functions are still being worked out, their deficiency has been termed "cerebellar cognitive affective syndrome" - a syndrome that bears a striking similarity to many of the symptoms of Alzheimer's disease (AD. Using ectopic cell cycle events and DNA damage markers as indexes of cellular distress, we have explored the neuropathological involvement of the CDN in human AD. Results We examined the human cerebellar dentate nucleus in 22 AD cases and 19 controls for the presence of neuronal cell cycle events and DNA damage using immunohistochemistry and fluorescence in situ hybridization. Both techniques revealed several instances of highly significant correlations. By contrast, neither amyloid plaque nor neurofibrillary tangle pathology was detected in this region, consistent with previous reports of human cerebellar pathology. Five cases of early stage AD were examined and while cell cycle and DNA damage markers were well advanced in the hippocampus of all five, few indicators of either cell cycle events (1 case or a DNA damage response (1 case were found in CDN. This implies that CDN neurons are most likely affected later in the course of AD. Clinical-pathological correlations revealed that cases with moderate to high levels of cell cycle activity in their CDN are highly likely to show deficits in unorthodox cerebellar functions including speech, language and motor planning. Conclusion Our results reveal that the CDN neurons are under cellular stress in AD and suggest that some of the non-motor symptoms found in patients with AD may be partly cerebellar in origin.

  18. Sequence specificity of alkali-labile DNA damage photosensitized by suprofen.

    Science.gov (United States)

    Starrs, S M; Davies, R J

    2000-09-01

    On irradiation at UVB wavelengths, in aerated neutral aqueous solution, the anti-inflammatory drug suprofen (SP) photosensitizes the production of alkali-labile cleavage sites in DNA much more efficiently than direct strand breaks. It is active at submillimolar concentrations despite having no significant binding affinity for DNA. Gel sequencing studies utilizing 32P-end-labeled oligonucleotides have revealed that piperidine-sensitive lesions are formed predominantly at the positions of guanine (G) bases, with the extent of modification being UV dose- and SP concentration-dependent. Quite distinct patterns of G-specific damage are observed in single-stranded and duplex DNA molecules. The uniform attack at all G residues in single-stranded DNA, which is enhanced in D2O, is compatible with a Type-II mechanism. SP is a known generator of singlet oxygen whose participation in the reaction is supported by the effects of quenchers and scavengers. In duplex DNA, piperidine-induced cleavage occurs with high selectivity at the 5'-G of GG and (less prominently) GA doublets. This behavior is characteristic of a Type-I process involving electron transfer from DNA to photoexcited SP molecules. The ability of SP to sensitize the formation of Type-I and Type-II photo-oxidation products from 2'-deoxyguanosine attests to the feasibility of competing mechanisms in DNA.

  19. How Does Thymine DNA Survive Ultrafast Dimerization Damage?

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    Hongjuan Wang

    2016-12-01

    Full Text Available The photodimerization reaction between the two adjacent thymine bases within a single strand has been the subject of numerous studies due to its potential to induce DNA mutagenesis and possible tumorigenesis in human skin cells. It is well established that the cycloaddition photoreaction takes place on a picosecond time scale along barrierless or low barrier singlet/triplet pathways. However, the observed dimerization quantum yield in different thymine multimer is considerable lower than might be expected. A reasonable explanation is required to understand why thymine in DNA is able to survive ultrafast dimerization damage. In this work, accurate quantum calculations based on the combined CASPT2//CASSCF/AMBER method were conducted to map the excited state relaxation pathways of the thymine monomer in aqueous solution and of the thymine oligomer in DNA. A monomer-like decay pathway, induced by the twisting of the methyl group, is found to provide a bypass channel to ensure the photostability of thymine in single-stranded oligomers. This fast relaxation path is regulated by the conical intersection between the bright SCT(1ππ* state with the intra-base charge transfer character and the ground state to remove the excess excitation energy, thereby achieving the ground-state recovery with high efficiency.

  20. Distinct roles of ATR and DNA-PKcs in triggering DNA damage responses in ATM-deficient cells

    Science.gov (United States)

    Tomimatsu, Nozomi; Mukherjee, Bipasha; Burma, Sandeep

    2009-01-01

    The cellular response to DNA double-strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell-cycle-dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell-cycle-independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM-deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM-deficient cells enforce an early G2/M checkpoint that is ATR-dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM-deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP-1), a protein involved in chromatin remodelling, is mediated by DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in a spatio-temporal manner in addition to ATM. We posit that ATM substrates involved in cell-cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA-PKcs in addition to ATM. PMID:19444312

  1. Irreparable telomeric DNA damage and persistent DDR signalling as a shared causative mechanism of cellular senescence and ageing.

    Science.gov (United States)

    Rossiello, Francesca; Herbig, Utz; Longhese, Maria Pia; Fumagalli, Marzia; d'Adda di Fagagna, Fabrizio

    2014-06-01

    The DNA damage response (DDR) orchestrates DNA repair and halts cell cycle. If damage is not resolved, cells can enter into an irreversible state of proliferative arrest called cellular senescence. Organismal ageing in mammals is associated with accumulation of markers of cellular senescence and DDR persistence at telomeres. Since the vast majority of the cells in mammals are non-proliferating, how do they age? Are telomeres involved? Also oncogene activation causes cellular senescence due to altered DNA replication and DDR activation in particular at the telomeres. Is there a common mechanism shared among apparently distinct types of cellular senescence? And what is the role of telomeric DNA damage? Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. IL-18 reduces ultraviolet radiation-induced DNA damage and thereby affects photoimmunosuppression.

    NARCIS (Netherlands)

    Schwarz, Agatha; Maeda, Akira; Ständer, Sonja; Steeg, Harry van; Schwarz, Thomas

    2006-01-01

    UV-induced DNA damage has been recognized as the major molecular trigger for photoimmunosuppression. IL-12 prevents UV-induced immunosuppression via its recently discovered capacity to reduce DNA damage presumably via induction of DNA repair. Because IL-18 shares some biological activities with

  3. Dynamics of the human nuclear proteome in response to DNA damage

    NARCIS (Netherlands)

    Dirksen, Eef Hubert Cecil

    2006-01-01

    The genome is constantly challenged by factors that can induce DNA damage and thereby threaten the viability of the cell. If DNA damage remains unrepaired it can lead to the development of cancer. Although much is known about the role of proteins and protein complexes in the cellular response to DNA

  4. 8-oxoG DNA Glycosylase-1 Inhibition Sensitizes Neuro-2a Cells to Oxidative DNA Base Damage Induced by 900 MHz Radiofrequency Electromagnetic Radiation

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    Xiaoya Wang

    2015-09-01

    Full Text Available Background/Aims: The purpose of this study was to explore the in vitro putative genotoxicity during exposure of Neuro-2a cells to radiofrequency electromagnetic fields (RF-EMFs with or without silencing of 8-oxoG DNA glycosylase-1 (OGG1. Methods: Neuro-2a cells treated with or without OGG1 siRNA were exposed to 900 MHz Global System for Mobile Communication (GSM Talk signals continuously at a specific absorption rate (SAR of 0, 0.5, 1 or 2 W/kg for 24 h. DNA strand breakage and DNA base damage were measured by the alkaline comet assay and a modified comet assay using formamidopyrimidine DNA glycosylase (FPG, respectively. Reactive oxygen species (ROS levels and cell viability were monitored using the non-fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA and CCK-8 assay. Results: Exposure to 900 MHz RF-EMFs with insufficient energy could induce oxidative DNA base damage in Neuro-2a cells. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS. Without OGG1 siRNA, 2 W/kg RF-EMFs induced oxidative DNA base damage in Neuro-2a cells. Interestingly, with OGG1 siRNA, RF-EMFs could cause DNA base damage in Neuro-2a cells as low as 1 W/kg. However, neither DNA strand breakage nor altered cell viability was observed. Conclusion: Even if further studies remain conducted we support the hypothesis that OGG1 is involved in the process of DNA base repair and may play a pivotal role in protecting DNA bases from RF-EMF induced oxidative damage.

  5. 8-oxoG DNA glycosylase-1 inhibition sensitizes Neuro-2a cells to oxidative DNA base damage induced by 900 MHz radiofrequency electromagnetic radiation.

    Science.gov (United States)

    Wang, Xiaoya; Liu, Chuan; Ma, Qinglong; Feng, Wei; Yang, Lingling; Lu, Yonghui; Zhou, Zhou; Yu, Zhengping; Li, Wei; Zhang, Lei

    2015-01-01

    The purpose of this study was to explore the in vitro putative genotoxicity during exposure of Neuro-2a cells to radiofrequency electromagnetic fields (RF-EMFs) with or without silencing of 8-oxoG DNA glycosylase-1 (OGG1). Neuro-2a cells treated with or without OGG1 siRNA were exposed to 900 MHz Global System for Mobile Communication (GSM) Talk signals continuously at a specific absorption rate (SAR) of 0, 0.5, 1 or 2 W/kg for 24 h. DNA strand breakage and DNA base damage were measured by the alkaline comet assay and a modified comet assay using formamidopyrimidine DNA glycosylase (FPG), respectively. Reactive oxygen species (ROS) levels and cell viability were monitored using the non-fluorescent probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) and CCK-8 assay. Exposure to 900 MHz RF-EMFs with insufficient energy could induce oxidative DNA base damage in Neuro-2a cells. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS). Without OGG1 siRNA, 2 W/kg RF-EMFs induced oxidative DNA base damage in Neuro-2a cells. Interestingly, with OGG1 siRNA, RF-EMFs could cause DNA base damage in Neuro-2a cells as low as 1 W/kg. However, neither DNA strand breakage nor altered cell viability was observed. Even if further studies remain conducted we support the hypothesis that OGG1 is involved in the process of DNA base repair and may play a pivotal role in protecting DNA bases from RF-EMF induced oxidative damage. © 2015 The Author(s) Published by S. Karger AG, Basel.

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

    Science.gov (United States)

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

    2000-01-01

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

  7. Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs

    International Nuclear Information System (INIS)

    Bounaix Morand du Puch, Ch

    2010-10-01

    DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

  8. Circular Mitochondrial DNA: A Geant4-DNA User Application for Evaluating Radiation-induced Damage in Circular Mitochondrial DNA.

    Science.gov (United States)

    Tavakoli, Mohammad Bagher; Moradi, Habiballah; Khanahmad, Hossein; Hosseini, Mohsen

    2017-01-01

    The aim of this study was to develop a nucleotide geometrical model of the circular mitochondrial DNA (mt-DNA) structure using Geant4-DNA toolkit to predict the radiation-induced damages such as single-strand breaks (SSB), double-strand breaks (DSB), and some other physical parameters. Our model covers the organization of a circular human mt genetic system. The current model includes all 16,659 base pairs of human mt-DNA. This new mt-DNA model has been preliminarily tested in this work by determining SSB and DSB DNA damage yields and site-hit probabilities due to the impact of proton particles. The accuracy of the geometry was determined by three-dimensional visualization in various ring element numbers. The hit locations were determined with respect to a reference coordinate system, and the corresponding base pairs were stored in the ROOT output file. The coordinate determination according to the algorithm was consistent with the expected results. The output results contain the information about the energy transfers in the backbone region of the DNA double helix. The output file was analyzed by root analyzing tools. Estimation of SSBs and DSBs yielded similar results with the increment of incident particle linear energy transfer. In addition, these values seem to be consistent with the corresponding experimental determinations. This model can be used in numerical simulations of mt-DNA radiation interactions to perform realistic evaluations of DNA-free radical reactions. This work will be extended to supercoiled conformation in the near future.

  9. Association of DNA damage and dyslipidemia with polycystic ovarian syndrome

    Directory of Open Access Journals (Sweden)

    Manikkumar R

    2013-02-01

    Full Text Available Polycystic ovary syndrome (PCOS is associated with hyperinsuli-nemia and insulin resistance which may lead to cardiovascular diseases. Evidence for cardiovascular events in women who were affected by PCOS during fertile age is limited. The pathogenesis is unknown; however, it is a complex multigenetic disorder. The present study was undertaken to evaluate the various cardiovas-cular risk factors and their DNA repair efficiency in women with PCOS by investigating the biochemical, endocrinological and mo-lecular cytogenetic alterations. These investigations were carried out in 116 women in the age group of 15-35 years clinically diag-nosed with PCOS. Data were compared with that of 50 age-matched healthy normal women. Fasting blood sugar (FBS, Lipid profile, Follicle-Stimulating Hormone (FSH and Luteinizing Hor-mone (LH, Prolactin and Estradiol were estimated after getting the informed consent. Mutagen induced chromosome sensitivity analysis was carried out in the lymphocytes of the subjects to as-sess the DNA repair proficiency. Fasting Blood Sugar, total cho-lesterol and LDL cholesterol were found to be elevated whereas HDL cholesterol was found to be lowered in the test subjects. FSH, LH and prolactin were also found to be significantly elevated in the test subjects. Change in the estradiol concentration in the test subjects was not significant. The mutagen sensitivity analysis revealed a significant elevation in break per cell (b/c values indi-cating a deficiency in the DNA repair mechanism / DNA damage in PCOS patients. Modification of life style by changing the dietary habit and sedentary life style will help to reduce the oxidative stress and may increase the ovarian function and a sensible life-style management is recommended for reducing the risk for CVD.

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

    Science.gov (United States)

    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.

  11. Cip29 is phosphorylated following activation of the DNA damage response in Xenopus egg extracts.

    Directory of Open Access Journals (Sweden)

    Janet Holden

    Full Text Available Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage.

  12. Detection, characterization and measure of a new radiation-induced damage in isolated and cellular DNA

    International Nuclear Information System (INIS)

    Regulus, P.

    2006-10-01

    Deoxyribonucleic acid (DNA) contains the genetic information and chemical injury to this macromolecule may have severe biological consequences. We report here the detection of 4 new radiation-induced DNA lesions by using a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) approach. For that purpose, the characteristic fragmentation of most 2'-deoxy-ribo nucleosides, the loss of 116 Da corresponding to the loss of the 2-deoxyribose moiety, was used in the so-called neutral loss mode of the HPLC-MS/MS. One of the newly detected lesions, named dCyd341 because it is a 2'-deoxycytidine modification exhibiting a molecular weight of 341 Da, was also detected in cellular DNA. Characterization of this modified nucleoside was performed using NMR and exact mass determination of the product obtained by chemical synthesis. A mechanism of formation was then proposed, in which the first event is the H-abstraction at the C4 position of a 2-deoxyribose moiety. Then, the sugar modification produced exhibits a reactive aldehyde that, through reaction with a vicinal cytosine base, gives rise to dCyd341. dCyd341 could be considered as a complex damage since its formation involves a DNA strand break and a cross-link between a damaged sugar residue and a vicinal cytosine base located most probably on the complementary DNA strand. In addition to its characterization, preliminary biological studies revealed that cells are able to remove the lesion from DNA. Repair studies have revealed the ability of cells to excise the lesion. Identification of the repair systems involved could represent an interesting challenge. (author)

  13. DNA damage and repair capacity in lymphocytes from obstructive sleep apnea patients.

    Science.gov (United States)

    Kontogianni, Konstantina; Messini-Nikolaki, Niki; Christou, Konstantinos; Gourgoulianis, Konstantinos; Tsilimigaki, Smaragdi; Piperakis, Stylianos M

    2007-12-01

    Obstructive sleep apnea (OSA) syndrome is a respiratory disease that is linked to heart attacks and high blood pressure. In the present study, we used the Comet assay to compare basal DNA damage and DNA damage induction by hydrogen peroxide, ethanol, and gamma-irradiation in lymphocytes from 35 OSA patients and 35 controls. We also measured the apoptosis and necrosis produced by these agents and the ability of the lymphocytes to repair the induced DNA damage. It was found that lymphocytes isolated from OSA patients had higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than lymphocytes from controls. OSA patients also had a reduced capacity to repair the DNA damage induced by the three agents, but apoptosis and necrosis were similar in OSA patients and the controls. (Copyright) 2007 Wiley-Liss, Inc.

  14. Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

    Science.gov (United States)

    Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

    2017-11-20

    DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

  15. Investigation of DNA damage and repair mechanism using deinococcus radiodurans

    International Nuclear Information System (INIS)

    Lau How Mooi; Kikuchi, M.; Kobayashi, Y.; Narumi, I.; Watanabe, H.

    1997-01-01

    Deninococcus Radiodurans, formerly known as Micrococcus Radiodurans, is a popular bacterium because of its high resistance to damage by carcinogens such as ionizing radiation (Dean et. al. 1966; Kitayama and Matsuyama 1968) and UV radiation (Gasvon et. al., 1995; Arrange et. al. 1993). In this report, we investigated the high resistance to ionizing radiation by this bacterium. The bacteria had been exposed from I to 5 kGy of gamma radiation and then incubated in TGY medium to study their ability to repair the broken DNA. The repair time was measured by Pulse Field Gel Electrophoresis (PFGE) method. The repair time for each dose was determined. Also in order to ensure that the repair was perfect, the bacterium was subjected to a second exposure of ionizing radiation after it has fully repaired. It was found that the 'second' repair characteristic was similar to the first repair. This confirmed that the repair after the exposure to the ionizing radiation was perfect

  16. Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

    Science.gov (United States)

    Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

    2012-11-09

    To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

  17. Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons*

    Science.gov (United States)

    Huelsenbeck, Stefanie C.; Schorr, Anne; Roos, Wynand P.; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

    2012-01-01

    To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons. PMID:23012366

  18. An alkaline separation method for detection of small amount of DNA damage

    International Nuclear Information System (INIS)

    Sakai, Kazuo; Okada, Shigefumi

    1981-01-01

    An alkaline separation technique originally established by Ahnstroem is modified to detect small amount of DNA damage in X-irradiated mouse leukemic L5178Y cells. It is made quantitative by calibration with an alkaline sucrose gradient centrifugation. The present method would make it possible to study DNA damage and its repair within a dose range of X-rays where cell survival and mutation are usually investigated. It is also useful for detecting DNA damage caused by chemicals. (author)

  19. Lead induced oxidative DNA damage in battery-recycling child workers from Bangladesh.

    Science.gov (United States)

    Arif, Mohammad; Islam, Mm Towhidul; Shekhar, Hossain Uddin

    2018-01-01

    Lead exposure can damage cells directly by effecting DNA or indirectly by modifying proteins and enzymes. In Bangladesh, many working children are exposed to a very high level of lead during their early life due to their involvement with lead-oriented professions. This imposes a severe threat to the growth and development of the children. Therefore to study the effect of lead, we enrolled 60 age-matched male children, from an area of old Dhaka city, where battery-recycling shops are located, depending on their blood lead concentration. If the children had a plasma lead concentration above the WHO recommended threshold level of 10 µg/dl, we grouped them as test subjects and others as control subjects to determine the effect of lead on different biochemical parameters of the body. Compared to the controls, acculumlation of the lipid peroxidation product, malondialdehyde, increased significantly in test subjects ( p < 0.01). Lead exposure also increased the protein carbonyl content ( p < 0.05) and significantly decreased the plasma glutathione levels of test subjects compared to the controls ( p < 0.05). While comparing the lead-exposed group against controls, it was found that the percentage of damaged DNA, as measured using the Comet assay, significantly increased in tail ( p < 0.01) and decreased in head regions. All of these results suggest that high-plasma lead content may induce an oxidative stress to the study population, which may lead to DNA damage.

  20. DNA damage in male gonad cells of Green mussel (Perna viridis) upon exposure to tobacco products

    Digital Repository Service at National Institute of Oceanography (India)

    Nagarajappa; Ganguly, A.; Goswami, U.

    , heavy metals, etc. Some of these chemicals are known genotoxicants and can cause damage to the genetic material of the exposed organism (Shugart, 1990). Damaged DNA may potentiate subsequent deleterious cellular events such as disease (e.g., cancer...

  1. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G., E-mail: lhudson@salud.unm.edu

    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.

  3. Oxidative stress and DNA damage in broad bean (Vicia faba L.) seedlings induced by thallium.

    Science.gov (United States)

    Radić, Sandra; Cvjetko, Petra; Glavas, Katarina; Roje, Vibor; Pevalek-Kozlina, Branka; Pavlica, Mirjana

    2009-01-01

    Thallium (Tl) is a metal of great toxicological concern because it is highly toxic to all living organisms through mechanisms that are yet poorly understood. Since Tl is accumulated by important crops, the present study aimed to analyze the biological effects induced by bioaccumulation of Tl in broad bean (Vicia faba L.) as well as the plant's antioxidative defense mechanisms usually activated by heavy metals. Thallium toxicity was related to production of reactive oxygen species in leaves and roots of broad bean seedlings following short-term (72 h) exposure to thallium (I) acetate (0, 0.5, 1, 5, and 10 mg/L) by evaluating DNA damage and oxidative stress parameters as well as antioxidative response. The possible antagonistic effect of potassium (K) was tested by combined treatment with 5 mg/L of Tl (Tl+) and 10 mg/L of potassium (K+) acetate. Accumulation of Tl+ in roots was 50 to 250 times higher than in broad bean shoots and was accompanied by increase in dry weight and proline. Despite responsive antioxidative defense (increased activities of superoxide dismutase, ascorbate peroxidase, and pyrogallol peroxidase), Tl+ caused oxidative damage to lipids and proteins as evaluated by malondialdehyde and carbonyl group levels, and induced DNA strand breaks. Combined treatment caused no oxidative alternations to lipids and proteins though it induced DNA damage. The difference in Tl-induced genotoxicity following both acellular and cellular exposure implies indirect DNA damage. Results obtained indicate that oxidative stress is involved in the mechanism of Tl toxicity and that the tolerance of broad bean to Tl is achieved, at least in part, through the increased activity of antioxidant enzymes.

  4. Poetry in motion: Increased chromosomal mobility after DNA damage.

    Science.gov (United States)

    Smith, Michael J; Rothstein, Rodney

    2017-08-01

    Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Balancing repair and tolerance of DNA damage caused by alkylating agents

    OpenAIRE

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D.

    2012-01-01

    Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial ...

  6. SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

    OpenAIRE

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

    2012-01-01

    Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial fo...

  7. Effect of low energy electron irradiation on DNA damage by Cu{sup 2+} ion

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Hyung Ah; Cho, Hyuck [Dept. of Physics, Chungnam National University, Daejeon (Korea, Republic of); Park, Yeun Soo [Plasma Technology Research Center, National Fusion Research Institute, Gunsan (Korea, Republic of)

    2017-03-15

    The combined effect of the low energy electron (LEE) irradiation and Cu{sup 2+} ion on DNA damage was investigated. Lyophilized pBR322 plasmid DNA films with various concentrations (1–15 mM) of Cu{sup 2+} ion were independently irradiated by monochromatic LEEs with 5 eV. The types of DNA damage, single strand break (SSB) and double strand break (DSB), were separated and quantified by gel electrophoresis. Without electron irradiation, DNA damage was slightly increased with increasing Cu ion concentration via Fenton reaction. LEE-induced DNA damage, with no Cu ion, was only 6.6% via dissociative electron attachment (DEA) process. However, DNA damage was significantly increased through the combined effect of LEE-irradiation and Cu ion, except around 9 mM Cu ion. The possible pathways of DNA damage for each of these different cases were suggested. The combined effect of LEE-irradiation and Cu ion is likely to cause increasing dissociation after elevated transient negative ion state, resulting in the enhanced DNA damage. For the decrease of DNA damage at around 9-mM Cu ion, it is assumed to be related to the structural stabilization due to DNA inter- and intra-crosslinks via Cu ion.

  8. Plasma Nuclear and Mitochondrial DNA Levels, and Markers of Inflammation, Shock, and Organ Damage in Patients with Septic Shock.

    Science.gov (United States)

    Timmermans, Kim; Kox, Matthijs; Scheffer, Gert Jan; Pickkers, Peter

    2016-06-01

    Plasma levels of the danger-associated molecular patterns (DAMPs) nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have been shown to be related to sepsis mortality. However, the intermediate factors and/or mechanisms contributing to this relation are largely unknown. Our aim was to determine whether plasma levels of nDNA and mtDNA are related to the markers of inflammation, severity of shock, and organ damage in septic shock patients. Moreover, we investigated the relationship between plasma levels of nDNA/mtDNA and inflammatory cytokines during experimental human endotoxemia, a model of systemic inflammation in humans in vivo mimicking some of the hallmarks of early sepsis. Blood was sampled from the onset of septic shock until day 28 in 121 septic shock patients and from 1 h before endotoxin administration until 8 h afterward in 12 healthy volunteers. Plasma concentrations of five cytokines and circulating levels of nDNA and mtDNA were measured, and correlations with shock-related parameters and markers of organ damage were investigated. In septic shock patients plasma cytokine concentrations, as well as nDNA and mtDNA levels, were increased at the onset of septic shock and remained elevated. During the first 5 days of septic shock, nDNA levels consistently correlated with plasma cytokine concentrations as well as with the shock-related parameter norepinephrine infusion rate and markers of organ damage (total bilirubin and creatinine). Experimental human endotoxemia also resulted in increased levels of plasma nDNA and mtDNA, but to a lesser extent than in septic shock patients. Furthermore, nDNA levels correlated with pro-inflammatory cytokines during endotoxemia. Our findings indicate a relationship between plasma nDNA levels and the inflammatory response. Furthermore, nDNA levels are associated with markers of shock and organ damage in septic shock patients. Nevertheless, the correlations found are relatively weak and it remains to be determined whether nDNA

  9. DNA damage in internal organs after cutaneous exposure to sulphur mustard

    Energy Technology Data Exchange (ETDEWEB)

    Batal, Mohamed [Laboratoire « Lésions des Acides Nucléiques », Université Joseph Fourier – Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France); Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP87, F-38702 La Tronche Cedex (France); Boudry, Isabelle; Mouret, Stéphane; Cléry-Barraud, Cécile; Wartelle, Julien [Département de Toxicologie et Risques Chimiques, Unité de Brûlure Chimique, Institut de Recherche Biomédicale des Armées, Antenne de La Tronche, BP87, F-38702 La Tronche Cedex (France); Bérard, Izabel [Laboratoire « Lésions des Acides Nucléiques », Université Joseph Fourier – Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France); Douki, Thierry, E-mail: thierry.douki@cea.fr [Laboratoire « Lésions des Acides Nucléiques », Université Joseph Fourier – Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble (France)

    2014-07-01

    Sulphur mustard (SM) is a chemical warfare agent that attacks mainly skin, eye and lungs. Due to its lipophilic properties, SM is also able to diffuse through the skin and reach internal organs. DNA represents one of the most critical molecular targets of this powerful alkylating agent which modifies DNA structure by forming monoadducts and biadducts. These DNA lesions are involved in the acute toxicity of SM as well as its long-term carcinogenicity. In the present work we studied the formation and persistence of guanine and adenine monoadducts and guanine biadducts in the DNA of brain, lungs, kidneys, spleen, and liver of SKH-1 mice cutaneously exposed to 2, 6 and 60 mg/kg of SM. SM-DNA adducts were detected in all studied organs, except in liver at the two lowest doses. Brain and lungs were the organs with the highest level of SM-DNA adducts, followed by kidney, spleen and liver. Monitoring the level of adducts for three weeks after cutaneous exposure showed that the lifetime of adducts were not the same in all organs, lungs being the organ with the longest persistence. Diffusion from skin to internal organs was much more efficient at the highest compared to the lowest dose investigated as the result of the loss of the skin barrier function. These data provide novel information on the distribution of SM in tissues following cutaneous exposures and indicate that brain is an important target. - Highlights: • Sulphur mustard reaches internal organs after skin exposure • Adducts are detected in the DNA of internal organs • Brain is the organ with the highest level of DNA damage • The barrier function of skin is lost at high dose of sulphur mustard • DNA adducts persist in organs for 2 or 3 weeks.

  10. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Science.gov (United States)

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

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

    Energy Technology Data Exchange (ETDEWEB)

    Srinivas, L.; Shalini, V.K. (Department of Nutrition and Food Safety, Central Food Technological Research Institute, Mysore (India))

    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.

  12. ANTIMUTAGENICITY OF CINNAMALDEHYDE AND VANILLIN IN HUMAN CELLS: GLOBAL GENE EXPRESSION AND POSSIBLE ROLE OF DNA DAMAGE AND REPAIR

    Science.gov (United States)

    This study investigated the possibility that chemicals identified as antimutagens may, in fact, operate through a mechanism involving DNA damage. We addressed this question by using two chemicals to which a large proportion of the population are exposed: vanillin and cinnemaldehy...

  13. TiO2 Photocatalysis Causes DNA Damage via Fenton Reaction-Generated Hydroxyl Radicals during the Recovery Period▿

    Science.gov (United States)

    Gogniat, Gaëtan; Dukan, Sam

    2007-01-01

    Here, we show that resistance of Escherichia coli to TiO2 photocatalysis involves defenses against reactive oxygen species. Results support the idea that TiO2 photocatalysis generates damage which later becomes deleterious during recovery. We found this to be partly due to DNA attack via hydroxyl radicals generated by the Fenton reaction during recovery. PMID:17933934

  14. DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Wyrobek, Andrew J.

    2009-01-18

    Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

  15. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Hammond, R.A.; Miller, M.R.; McClung, J.K.

    1990-01-01

    The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

  16. Hormone-induced DNA damage response and repair mediated by cyclin D1 in breast and prostate cancer.

    Science.gov (United States)

    Di Sante, Gabriele; Di Rocco, Agnese; Pupo, Claudia; Casimiro, Mathew C; Pestell, Richard G

    2017-10-10

    Cell cycle control proteins govern events that leads to the production of two identical daughter cells. Distinct sequential temporal phases, Gap 1 (G 1 ), Gap 0 (G 0 ), Synthesis (S), Gap 2 (G 2 ) and Mitosis (M) are negotiated through a series of check points during which the favorability of the local cellular environment is assessed, prior to replicating DNA [1]. Cyclin D1 has been characterized as a key regulatory subunit of the holoenzyme that promotes the G 1 /S-phase transition through phosphorylating the pRB protein. Cyclin D1 overexpression is considered a driving force in several types of cancers and cdk inhibitors are being used effectively in the clinic for treatment of ERα + breast cancer [1, 2]. Genomic DNA is assaulted by damaging ionizing radiation, chemical carcinogens, and reactive oxygen species (ROS) which are generated by cellular metabolism. Furthermore, specific hormones including estrogens [3, 4] and androgens [5] govern pathways that damage DNA. Defects in the DNA Damage Response (DDR) pathway can lead to genomic instability and cancer. Evidence is emerging that cyclin D1 bind proteins involved in DNA repair including BRCA1 [6], RAD51 [7], BRCA2 [8] and is involved in the DNA damage and DNA repair processes [7, 8]. Because the repair of damaged DNA appears to be an important and unexpected role for cyclin D1, and inhibitors of cyclin D1-dependent kinase activity are being used in the clinic, the latest findings on the role of cyclin D1 in mediating the DDR including the DDR induced by the hormones estrogen [9] and androgen [10, 11] is reviewed.

  17. Host DNA damage response factors localize to merkel cell polyomavirus DNA replication sites to support efficient viral DNA replication.

    Science.gov (United States)

    Tsang, Sabrina H; Wang, Xin; Li, Jing; Buck, Christopher B; You, Jianxin

    2014-03-01

    Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR-mediated DDR

  18. Participation of ATM in cellular response to DNA damage induced by ionizing radiation

    International Nuclear Information System (INIS)

    Meng Xiangbing; Song Yi; Mao Jianping; Gong Bo; Dong Yan; Liu Bin; Sun Zhixian

    2000-01-01

    Objective: To clone ATM full length cDNA and cDNA fragments containing some functional domains and to identify proteins that interact with ATM and mediate DNA damage signal transduction in cellular response to DNA damage. Methods: ATM cDNA was amplified from MarthomTM-Ready cDNA kit of human leukocytes by LD-PCR. ATM-interacting proteins were screened by yeast two hybrid system. Results: ATM full-length cDNA and cDNA fragments containing PI3K kinase domain, leucine zipper and proline rich region were amplified from human cDNAs. Several candidate clones that interacted with ATM PI3K domain were identified. Conclusion: ATM mediates DNA damage signal transduction by interacting with many proteins

  19. A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation

    Directory of Open Access Journals (Sweden)

    Kumar Sonia

    2011-02-01

    Full Text Available Abstract Background The mammalian DNA-damage response (DDR has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs, mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. Methods In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+ with the MDM2 antagonist, Nutlin-3. Results Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37. Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. Conclusion To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

  20. DNA Damage Induction and Repair Evaluated in Human Lymphocytes Irradiated with X-Rays an Neutrons

    International Nuclear Information System (INIS)

    Niedzwiedz, W.; Cebulska-Wasilewska, A.

    2000-12-01

    The objective of this study was to evaluate the kinetic of the DNA damage induction and their subsequent repair in human lymphocytes exposed to various types of radiation. PBLs cells were isolated from the whole blood of two young healthy male subjects and one skin cancer patient, and than exposed to various doses of low LET X-rays and high LET neutrons from 252 Cf source. To evaluate the DNA damage we have applied the single cell get electrophoresis technique (SCGE) also known as the comet assay. In order to estimate the repair efficiency, cells, which had been irradiated with a certain dose, were incubated at 37 o C for various periods of time (0 to 60 min). The kinetic of DNA damage recovery was investigated by an estimation of residual DNA damage persisted at cells after various times of post-irradiation incubation (5, 10, 15, 30 and 60 min). We observed an increase of the DNA damage (reported as a Tail DNA and Tail moment parameters) in linear and linear-quadratic manner, with increasing doses of X-rays and 252 Cf neutrons, respectively. Moreover, for skin cancer patient (Code 3) at whole studied dose ranges the higher level of the DNA damage was observed comparing to health subjects (Code 1 and 2), however statistically insignificant (for Tail DNA p=0.056; for Tail moment p=0.065). In case of the efficiency of the DNA damage repair it was observed that after 1 h of post-irradiation incubation the DNA damage induced with both, neutrons and X-rays had been significantly reduced (from 65% to 100 %). Furthermore, in case of skin cancer patient we observed lover repair efficiency of X-rays induced DNA damage. After irradiation with neutrons within first 30 min, the Tail DNA and Tail moment decreased of about 50%. One hour after irradiation, almost 70% of residual and new formed DNA damage was still observed. In this case, the level of unrepaired DNA damage may represent the fraction of the double strand breaks as well as more complex DNA damage (i.e.-DNA or DNA

  1. DNA-Damage-Induced Type I Interferon Promotes Senescence and Inhibits Stem Cell Function

    Directory of Open Access Journals (Sweden)

    Qiujing Yu

    2015-05-01

    Full Text Available Expression of type I interferons (IFNs can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

  2. Estrogen signalling and the DNA damage response in hormone dependent breast cancers

    Directory of Open Access Journals (Sweden)

    C Elizabeth Caldon

    2014-05-01

    Full Text Available Estrogen is necessary for the normal growth and development of breast tissue, but high levels of estrogen are a major risk factor for breast cancer. One mechanism by which estrogen could contribute to breast cancer is via the induction of DNA damage. This perspective discusses the mechanisms by which estrogen alters the DNA damage response (DDR and DNA repair through the regulation of key effector proteins including ATM, ATR, CHK1, BRCA1 and p53 and the feedback on estrogen receptor signalling from these proteins. We put forward the hypothesis that estrogen receptor signalling converges to suppress effective DNA repair and apoptosis in favour of proliferation. This is important in hormone-dependent breast cancer as it will affect processing of estrogen-induced DNA damage, as well as other genotoxic insults. DDR and DNA repair proteins are frequently mutated or altered in estrogen responsive breast cancer which will further change the processing of DNA damage. Finally the action of estrogen signalling on DNA damage is also relevant to the therapeutic setting as the suppression of a DNA damage response by estrogen has the potential to alter the response of cancers to anti-hormone treatment or chemotherapy that induces DNA damage.

  3. Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay

    Energy Technology Data Exchange (ETDEWEB)

    Wakizaka, A.; Nishizawa, Y.; Aiba, N.; Okuhara, E.; Takahashi, S.

    1989-02-01

    A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells.

  4. Quantitative analysis of gene-specific DNA damage in human spermatozoa

    International Nuclear Information System (INIS)

    Sawyer, Dennis E.; Mercer, Belinda G.; Wiklendt, Agnieszka M.; Aitken, R. John

    2003-01-01

    Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H 2 O 2 ; 0-5 mM) or iron (as Fe(II)SO 4 , 0-500 μM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, β-pol and β-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H 2 O 2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H 2 O 2 . The mitochondrial genome of human spermatozoa was significantly (P 2 O 2 -induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage

  5. Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Directory of Open Access Journals (Sweden)

    Eveson J Paige

    2006-08-01

    Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

  6. HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

    Science.gov (United States)

    Ito, Hikaru; Fujita, Kyota; Tagawa, Kazuhiko; Chen, Xigui; Homma, Hidenori; Sasabe, Toshikazu; Shimizu, Jun; Shimizu, Shigeomi; Tamura, Takuya; Muramatsu, Shin-ichi; Okazawa, Hitoshi

    2015-01-01

    Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset. PMID:25510912

  7. In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

    Czech Academy of Sciences Publication Activity Database

    Villani, G.; Hübscher, U.; Gironis, N.; Parkkinen, S.; Pospiech, H.; Shevelev, Igor; di Cicco, G.; Markennen, E.; Syvaaja, J.E.; Le Gac, N.T.

    2011-01-01

    Roč. 286, č. 37 (2011), s. 32094-32104 ISSN 0021-9258 Grant - others:Academy of Finland(FI) 106986; Academy of Finland(FI) 123082 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA damage * DNA polymerase * DNA repair * DNA replication * DNA-protein interaction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.773, year: 2011

  8. A novel function of CRL4(Cdt2): regulation of the subunit structure of DNA polymerase δ in response to DNA damage and during the S phase.

    Science.gov (United States)

    Zhang, Sufang; Zhao, Hong; Darzynkiewicz, Zbiegniew; Zhou, Pengbo; Zhang, Zhongtao; Lee, Ernest Y C; Lee, Marietta Y W T

    2013-10-11

    DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4(Cdt2), a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4(Cdt2) included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4(Cdt2) complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4(Cdt2) also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4(Cdt2), i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair.

  9. Oxidative DNA damage in vitamin C-supplemented guinea pigs after intratracheal instillation of diesel exhaust particles

    DEFF Research Database (Denmark)

    Moller, P.; Daneshvar, B.; Loft, S.

    2003-01-01

    The health effects of diesel exhaust particles (DEP) are thought to involve oxidative damage. We have investigated the effect of intratracheal DEP instillation to guinea pigs in three groups of 12 animals each given 0, 0.7, or 2.1 mg. Five days later guinea pigs exposed to DEP had increased levels...... of oxidized amino acids (gamma-glutamyl semialdehyde), DNA strand breaks, and 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in the lung. Bulky DNA ad- ducts were not significantly elevated in the lung. The antioxidant enzyme activity of glutathione reductase was increased in the lung of DEP-exposed guinea pigs....... The concentrations of ascorbate in liver, lung, and plasma were unaltered by the DEP exposure. The results indicate that in guinea pigs DEP causes oxidative DNA damage rather than bulky DNA adducts in the lung. Guinea pigs, which are similar to humans with respect to vitamin C metabolism, may serve as a new model...

  10. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass

    DEFF Research Database (Denmark)

    Povlsen, Lou K; Beli, Petra; Wagner, Sebastian A

    2012-01-01

    to mono-ubiquitylated PCNA at stalled replisomes. Our findings demonstrate widespread involvement of ubiquitin signalling in genotoxic-stress responses and identify a critical function for dynamic PAF15 ubiquitylation in safeguarding genome integrity when DNA replication is challenged.......Protein ubiquitylation has emerged as a key regulatory mechanism in DNA-damage signalling and repair pathways. We report a proteome-wide, site-specific survey of ubiquitylation changes after ultraviolet irradiation, identifying numerous upregulated and downregulated ubiquitylation sites on known...... components of DNA-damage signalling, as well as on proteins not previously implicated in this process. Our results uncover a critical role for PCNA-associated factor PAF15 (p15(PAF)/KIAA0101) ubiquitylation during DNA replication. During unperturbed S phase, chromatin-associated PAF15 is modified by double...

  11. Method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin

    International Nuclear Information System (INIS)

    Singh, G.; Hauswirth, W.W.; Ross, W.E.; Neims, A.H.

    1985-01-01

    This paper describes a rapid and reliable method for quantification of damage to mitochondrial DNA (mtDNA), especially strand breaks. The degree of damage to mtDNA is assessed by the proportion of physical forms (i.e., supercoiled versus open-circular and linear forms) upon agarose gel electrophoresis, blotting, and visualization by hybridization with [ 32 P]mtDNA probes. The use of a radiolabeled probe is a crucial step in the procedure because it provides both a means to quantify by radioautography and to obtain the mtDNA specificity required to eliminate misinterpretation due to nuclear DNA contamination. To demonstrate the utility of this technique, X-irradiation and epichlorohydrin are shown to damage both isolated mtDNA and mtDNA in whole cells in a dose-dependent fashion

  12. Protective effect of antioxidants on DNA damage in leukocytes from X-linked adrenoleukodystrophy patients.

    Science.gov (United States)

    Marchetti, Desirèe P; Donida, Bruna; da Rosa, Helen T; Manini, Paula R; Moura, Dinara J; Saffi, Jenifer; Deon, Marion; Mescka, Caroline P; Coelho, Daniella M; Jardim, Laura B; Vargas, Carmen R

    2015-06-01

    Toxic metabolites accumulation and oxidative stress have been associated to the pathophysiology of X-linked adrenoleukodystrophy (X-ALD), an inborn error of peroxisome metabolism. Parameters of oxidative damage to proteins and lipids in X-ALD patients were already described in literature; however, DNA injuries were not studied yet. Considering that, the aims were to investigate DNA damage by comet assay in heterozygotes and symptomatic X-ALD patients, to look for associations between DNA damage and lipid peroxidation as measured by urinary 15-F2t-isoprostane; and to evaluate the in vitro effect of N-acetyl-l-cysteine (NAC), trolox (TRO) and rosuvastatin (RSV) on DNA damage in leukocytes from symptomatic patients. Symptomatic patients presented higher DNA damage levels than those found in heterozygotes and controls; heterozygotes and controls showed similar results. In order to investigate the in vitro antioxidant effect on DNA damage, whole blood cells from symptomatic patients were incubated with NAC (1 and 2.5mM), TRO (25 and 75 μM) and RSV (0.5, 2 and 5 μM) before DNA damage analysis. NAC, TRO and RSV, at all tested concentrations, were all capable to reduce DNA damage in symptomatic X-ALD patients until control levels. Finally, DNA damage correlated with urinary isoprostanes and plasmatic levels of TBA-RS and DCFH-DA, allowing to hypothesize that DNA damage might be induced by lipid peroxidation in symptomatic patients. The present work yields experimental evidence that NAC, TRO and RSV reduce the in vitro DNA injury in symptomatic X-ALD patients, what may suggest that the administration of these antioxidants might be considered as an adjuvant therapy for X-ALD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Inter-laboratory variation in DNA damage using a standard comet assay protocol

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Ersson, Clara; Loft, Steffen

    2012-01-01

    There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories ...

  14. Associations of subjective vitality with DNA damage, cardiovascular risk factors and physical performance

    DEFF Research Database (Denmark)

    Maynard, Scott; Keijzers, Guido; Hansen, A-M

    2015-01-01

    To examine associations of DNA damage, cardiovascular risk factors and physical performance with vitality, in middle-aged men. We also sought to elucidate underlying factors of physical performance by comparing physical performance parameters to DNA damage parameters and cardiovascular risk factors....

  15. Bidirectional coupling of splicing and ATM signaling in response to transcription-blocking DNA damage

    NARCIS (Netherlands)

    M. Tresini (Maria); J.A. Marteijn (Jurgen); W. Vermeulen (Wim)

    2016-01-01

    textabstractIn response to DNA damage cells activate intricate protein networks to ensure genomic fidelity and tissue homeostasis. DNA damage response signaling pathways coordinate these networks and determine cellular fates, in part, by modulating RNA metabolism. Here we discuss a

  16. DNA-damage response during mitosis induces whole-chromosome missegregation.

    Science.gov (United States)

    Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A

    2014-11-01

    Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

  17. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE: INDUCED BY RADIATION, CHEMICALS AND ENZYMES

    Science.gov (United States)

    A simple and rapid assay to detect DNA damage is reported. This assay is based on the ability of certain dyes to fluoresce upon intercalation with dsDNA. Damage caused by ultraviolet (UV) radiation, chemicals or restriction enzymes is detected using this assay. UV radiation at...

  18. DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation.

    Directory of Open Access Journals (Sweden)

    Andreas Lamkowski

    Full Text Available Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs. The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI with 49 Gy (± 6% Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available

  19. Alpha, beta-unsaturated lactones 2-furanone and 2-pyrone induce cellular DNA damage, formation of topoisomerase I- and II-DNA complexes and cancer cell death.

    Science.gov (United States)

    Calderón-Montaño, José Manuel; Burgos-Morón, Estefanía; Orta, Manuel Luis; Pastor, Nuria; Austin, Caroline A; Mateos, Santiago; López-Lázaro, Miguel

    2013-09-12

    The alpha, beta-unsaturated lactones 2-furanone and 2-pyrone are part of the chemical structure of a variety of naturally occurring compounds (e.g., cardenolides, bufadienolides, acetogenins, coumarins, and food-flavoring furanones), some of which have shown anticancer activity and/or DNA damaging effects. Here we report that 2-furanone and 2-pyrone induce cellular DNA damage (assessed by the comet assay and the gamma-H2AX focus assay) and the formation of topoisomerase I- and topoisomerase II-DNA complexes in cells (visualized and quantified in situ by the TARDIS assay). Cells mutated in BRCA2 (deficient in homologous recombination repair) were significantly hypersensitive to the cytotoxic activity of 2-pyrone, therefore suggesting that BRCA2 plays an important role in the repair of DNA damage induced by this lactone. Both lactones were cytotoxic in A549 lung cancer cells at lower concentrations than in MRC5 non-malignant lung fibroblasts. The possible involvement of 2-furanone and 2-pyrone in the anticancer and DNA-damaging activities of compounds containing these lactones is discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. Colorimetric detection of DNA damage by using hemin-graphene nanocomposites

    Science.gov (United States)

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

    2013-04-01

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

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

    Science.gov (United States)

    Plante, Ianik; Cicinotta, Francis A.

    2013-01-01

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

  2. Multi-scale approach to radiation damage induced by ion beams: complex DNA damage and effects of thermal spikes

    International Nuclear Information System (INIS)

    Surdutovich, E.; Yakubovich, A.V.; Solov'yov, A.V.; Surdutovich, E.; Yakubovich, A.V.; Solov'yov, A.V.

    2010-01-01

    We present the latest advances of the multi-scale approach to radiation damage caused by irradiation of a tissue with energetic ions and report the calculations of complex DNA damage and the effects of thermal spikes on biomolecules. The multi-scale approach aims to quantify the most important physical, chemical, and biological phenomena taking place during and following irradiation with ions and provide a better means for clinically-necessary calculations with adequate accuracy. We suggest a way of quantifying the complex clustered damage, one of the most important features of the radiation damage caused by ions. This quantification allows the studying of how the clusterization of DNA lesions affects the lethality of damage. We discuss the first results of molecular dynamics simulations of ubiquitin in the environment of thermal spikes, predicted to occur in tissue for a short time after an ion's passage in the vicinity of the ions' tracks. (authors)

  3. Oxidative stress and inflammation generated DNA damage by exposure to air pollution particles

    DEFF Research Database (Denmark)

    Møller, Peter; Danielsen, Pernille Høgh; Karottki, Dorina Gabriela

    2014-01-01

    investigating air pollution particles. There is substantial evidence indicating that exposure to air pollution particles was associated with elevated levels of oxidatively damaged nucleobases in circulating blood cells and urine from humans, which is supported by observations of elevated levels of genotoxicity...... of PM sampled at different locations or times. Small air pollution particles did not appear more hazardous than larger particles, which is consistent with the notion that constituents such as metals and organic compounds also are important determinants for PM-generated oxidative stress and inflammation....... In addition, the results indicate that PM-mediated ROS production is involved in the generation of inflammation and activated inflammatory cells can increase their ROS production. The observations indicate that air pollution particles generate oxidatively damaged DNA by promoting a milieu of oxidative stress...

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

    Energy Technology Data Exchange (ETDEWEB)

    Kiran, Shashi; Oddi, Vineesha [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Ramakrishna, Gayatri, E-mail: gayatrirama1@gmail.com [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences, Delhi 110070 (India)

    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

  5. A Real-Time QCM-D Approach to Monitoring Mammalian DNA Damage Using DNA Adsorbed to a Polyelectrolyte Surface

    Science.gov (United States)

    Rawle, Robert J.; Johal, Malkiat S.; Selassie, Cynthia R. D.

    2008-01-01

    We have successfully demonstrated that the quartz crystal microbalance with dissipation monitoring (QCM-D) can be used to monitor real-time damage to genomic mammalian DNA adsorbed to a polyelectrolyte surface. To reveal the capabilities of this technique, we exposed DNA surfaces to quercetin, an agent that has been implicated in causing DNA strand breaks in a Cu(II)-dependent fashion in vitro. We show that the QCM-D frequency and dissipation patterns that result from exposure of the DNA surfaces to quercetin/Cu(II) are consistent with the induction of DNA strand scission. We use QCM-D to furthermore demonstrate that this process is dependent on Cu(II) and that the DNA damage induced by quercetin can still be detected if Cu(II) is in situ with the DNA surface and not in solution-phase. PMID:18076139

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

    Science.gov (United States)

    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.

  7. Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage

    International Nuclear Information System (INIS)

    Stapleton, A.E.; Walbot, V.

    1994-01-01

    Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage. (author)

  8. Accumulation of lipids and oxidatively damaged DNA in hepatocytes exposed to particles

    DEFF Research Database (Denmark)

    Vesterdal, Lise K; Danielsen, Pernille H; Folkmann, Janne K

    2014-01-01

    and subsequently incubated for another 18h to manifest lipid accumulation. In an animal model of metabolic syndrome we investigated the association between intake of carbon black (CB, 14nm) particles and hepatic lipid accumulation, inflammation and gene expression of Srebp-1, Fasn and Scd-1 involved in lipid...... single-walled carbon nanotubes. All four types of particles also generated oxidatively damaged DNA, assessed as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, in HepG2 cells after 3h exposure. The animal model of metabolic syndrome showed increased lipid load in the liver after one oral......Exposure to particles has been suggested to generate hepatosteatosis by oxidative stress mechanisms. We investigated lipid accumulation in cultured human hepatocytes (HepG2) and rat liver after exposure to four different carbon-based particles. HepG2 cells were exposed to particles for 3h...

  9. Deregulation upon DNA damage revealed by joint analysis of context-specific perturbation data

    Directory of Open Access Journals (Sweden)

    Biecek Przemysław

    2011-06-01

    Full Text Available Abstract Background Deregulation between two different cell populations manifests itself in changing gene expression patterns and changing regulatory interactions. Accumulating knowledge about biological networks creates an opportunity to study these changes in their cellular context. Results We analyze re-wiring of regulatory networks based on cell population-specific perturbation data and knowledge about signaling pathways and their target genes. We quantify deregulation by merging regulatory signal from the two cell populations into one score. This joint approach, called JODA, proves advantageous over separate analysis of the cell populations and analysis without incorporation of knowledge. JODA is implemented and freely available in a Bioconductor package 'joda'. Conclusions Using JODA, we show wide-spread re-wiring of gene regulatory networks upon neocarzinostatin-induced DNA damage in Human cells. We recover 645 deregulated genes in thirteen functional clusters performing the rich program of response to damage. We find that the clusters contain many previously characterized neocarzinostatin target genes. We investigate connectivity between those genes, explaining their cooperation in performing the common functions. We review genes with the most extreme deregulation scores, reporting their involvement in response to DNA damage. Finally, we investigate the indirect impact of the ATM pathway on the deregulated genes, and build a hypothetical hierarchy of direct regulation. These results prove that JODA is a step forward to a systems level, mechanistic understanding of changes in gene regulation between different cell populations.

  10. Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans

    Directory of Open Access Journals (Sweden)

    Diletta Edifizi

    2017-08-01

    Full Text Available DNA damage causally contributes to aging and age-related diseases. Mutations in nucleotide excision repair (NER genes cause highly complex congenital syndromes characterized by growth retardation, cancer susceptibility, and accelerated aging in humans. Orthologous mutations in Caenorhabditis elegans lead to growth delay, genome instability, and accelerated functional decline, thus allowing investigation of the consequences of persistent DNA damage during development and aging in a simple metazoan model. Here, we conducted proteome, lipidome, and phosphoproteome analysis of NER-deficient animals in response to UV treatment to gain comprehensive insights into the full range of physiological adaptations to unrepaired DNA damage. We derive metabolic changes indicative of a tissue maintenance program and implicate an autophagy-mediated proteostatic response. We assign central roles for the insulin-, EGF-, and AMPK-like signaling pathways in orchestrating the adaptive response to DNA damage. Our results provide insights into the DNA damage responses in the organismal context.

  11. BRCA1 in the DNA damage response and at telomeres

    Directory of Open Access Journals (Sweden)

    Eliot Michael Rosen

    2013-06-01

    Full Text Available Abstract. Mutations of the breast and ovarian cancer susceptibility gene 1 (BRCA1 account for about 40-45% of hereditary breast cancer cases. Moreover, a significant fraction of sporadic (non-hereditary breast and ovarian cancers exhibit reduced or absent expression of the BRCA1 protein, suggesting an additional role for BRCA1 in sporadic cancers. BRCA1 follows the classic pattern of a highly penetrant Knudsen-type tumor suppressor gene in which one allele is inactivated through a germ-line mutation and the other is mutated or deleted within the tumor. BRCA1 is a multi-functional protein but it is not fully understood which function(s is (are most important for tumor suppression, nor is it clear why BRCA1 mutations confer a high risk for breast and ovarian cancers and not a broad spectrum of tumor types. Here, we will review BRCA1 functions in the DNA damage response (DDR, which are likely to contribute to tumor suppression. In the process, we will highlight some of the controversies and unresolved issues in the field. We will also describe a recently identified and under-investigated role for BRCA1 in the regulation of telomeres and the implications of this role in the DDR and cancer suppression.

  12. Personal exposure to ultrafine particles and oxidative DNA damage

    DEFF Research Database (Denmark)

    Vinzents, Peter S; Møller, Peter; Sørensen, Mette

    2005-01-01

    Exposure to ultrafine particles (UFPs) from vehicle exhaust has been related to risk of cardiovascular and pulmonary disease and cancer, even though exposure assessment is difficult. We studied personal exposure in terms of number concentrations of UFPs in the breathing zone, using portable instr......, particularly during bicycling in traffic. The results indicate that biologic effects of UFPs occur at modest exposure, such as that occurring in traffic, which supports the relationship of UFPs and the adverse health effects of air pollution.......Exposure to ultrafine particles (UFPs) from vehicle exhaust has been related to risk of cardiovascular and pulmonary disease and cancer, even though exposure assessment is difficult. We studied personal exposure in terms of number concentrations of UFPs in the breathing zone, using portable...... instruments in six 18-hr periods in 15 healthy nonsmoking subjects. Exposure contrasts of outdoor pollution were achieved by bicycling in traffic for 5 days and in the laboratory for 1 day. Oxidative DNA damage was assessed as strand breaks and oxidized purines in mononuclear cells isolated from venous blood...

  13. 2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

    International Nuclear Information System (INIS)

    El-Yazbi, Amira F.; Loppnow, Glen R.

    2012-01-01

    Highlights: ► Molecular beacon with 2AP bases detects DNA damage in a simple mix-and-read assay. ► Molecular beacons with 2AP bases detect damage at a 17.2 nM limit of detection. ► The 2AP molecular beacon is linear over a 0–3.5 μM concentration range for damage. - Abstract: Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R 2 = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.

  14. Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.

    Science.gov (United States)

    Li, Hui; Swiercz, Rafal; Englander, Ella W

    2009-09-01

    Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death.

  15. Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron-overload in the brain on integrity of neuronal DNA

    Science.gov (United States)

    Li, Hui; Swiercz, Rafal; Englander, Ella W.

    2009-01-01

    Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Since in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5’-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations, which inhibit BER activities in in vitro assays, do not induce direct genomic damage in cultured primary neurons, they significantly delay repair of genomic DNA damage induced by sub-lethal exposure to H2O2. Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death. PMID:19619136

  16. Repair of radiation-induced DNA damage in rat epidermis as a function of age

    International Nuclear Information System (INIS)

    Sargent, E.V.; Burns, F.J.

    1985-01-01

    The rate of repair of radiation-induced DNA damage in proliferating rat epidermal cells diminished progressively with increasing age of the animal. The dorsal skin was irradiated with 1200 rad of 0.8 MeV electrons at various ages, and the amount of DNA damage was determined as a function of time after irradiation by the method of alkaline unwinding followed by S 1 nuclease digestion. The amount of DNA damage immediately after irradiation was not age dependent, while the rate of damage removal from the DNA decreased with increasing age. By fitting an exponential function to the relative amount of undamaged DNA as a function of time after irradiation, DNA repair halftimes of 20, 27, 69, and 107 min were obtained for 28, 100-, 200-, and 400-day-old animals, respectively

  17. DNA damage in lung after oral exposure to diesel exhaust particles in Big Blue (R) rats

    DEFF Research Database (Denmark)

    Müller, Anne Kirstine; Farombi, E.O.; Møller, P.

    2004-01-01

    Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts....... Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated......, in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue(R) rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks...

  18. DNA Damage Is a Potential Marker for TP53 Mutation in Colorectal Carcinogenesis.

    Science.gov (United States)

    Scalise, José Ricardo; Poças, Regina Caeli Guerra; Caneloi, Thamy Pelatieri; Lopes, Camila Oliveira; Kanno, Danilo Toshio; Marques, Mayara Gonçalves; Valdivia, Júlio Cesar Martins; Maximo, Felipe Rodrigues; Pereira, José Aires; Ribeiro, Marcelo Lima; Priolli, Denise Gonçalves

    2016-12-01

    The ability to measure oxidative DNA damage in a tissue allows establishment of the relationship between DNA damage and mutations in normal and neoplastic cells. It is well known that TP53 is a key inhibitor of tumor development and preserves the genome integrity in each cell. The aim of the present study was to investigate the relationship between DNA damage and TP53 mutation in colorectal adenoma and adenocarcinoma, and the value of DNA damage as potential marker of TP53 mutation in non-tumor tissues adjacent to colon malignant lesions. Tissue samples were obtained by colonoscopy from patients with adenoma and/or adenocarcinoma and from healthy volunteers. Diagnosis was defined by histopathology. Immunohistochemistry with computer-assisted image analysis was performed to quantify TP53 mutation. Oxidative DNA damage was determined by comet assay. Statistical analyses were performed with 5 % of significance level. The TP53 level was higher in non-tumor tissues from tumor patients than in normal tissues from healthy volunteers (p = 0.01). Likewise, higher TP53 levels were observed in tumor tissues compared with the non-tumor tissues (p = 0.00). Oxidative DNA damage levels were higher in tumor tissues than in non-tumor tissues (p = 0.00). The amount of TP53 (p = 0.00) and oxidative DNA damage (p = 0.00) in normal and tumor tissue was related. The relationship between oxidative DNA damage and TP53 mutation was demonstrated in all samples (p = 0.00). Oxidative DNA damage is an intervening variable for TP53 mutation in colorectal adenoma-carcinoma. Our data suggests that oxidative DNA damage is a potential marker of TP53 mutation in colorectal carcinogenesis.

  19. ShaPINg cell fate upon DNA damage:role of Pin1 isomerase in DNA damage-induced cell death and repair

    Directory of Open Access Journals (Sweden)

    Thomas G Hofmann

    2014-06-01

    Full Text Available The peptidyl-prolyl cis/trans isomerase Pin1 acts as a molecular timer in proline-directed Ser/Thr kinase signaling and shapes cellular responses based on recognition of phosphorylation marks and implementing conformational changes in its substrates. Accordingly, Pin1 has been linked to numerous phosphorylation-controlled signaling pathways and cellular processes such as cell cycle progression, proliferation and differentiation. In addition, Pin1 plays a pivotal role in DNA damage-triggered cell fate decisions. Whereas moderate DNA damage is balanced by DNA repair, cells confronted with massive genotoxic stress are eliminated by the induction of programmed cell death or cellular senescence. In this review we summarize and discuss the current knowledge on how Pin1 specifies cell fate through regulating key players of the apoptotic and the repair branch of the DNA damage response.

  20. DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis

    OpenAIRE

    Durbach, S I; Springer, B; Machowski, E E; North, R J; Papavinasasundaram, K G; Colston, M J; Böttger, E C; Mizrahi, V

    2003-01-01

    One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is res...

  1. Natural transformation of bacteria by fragmented, damaged and ancient DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    Organisms release DNA both when they live and die. Eventually the DNA disintegrates entirely or it is re-metabolized. There is a constant deposition and decomposition that maintains an environmental pool with large quantities of extracellular DNA, some of which can be thousands of years old. The ...... in chemistry. In addition to qualitative DNA sequencing, statistical analysis of the numerous sequencing reads yields quantitative measurements of DNA modifying reactions....

  2. DNA damage in lung after oral exposure to diesel exhaust particles in Big Blue (R) rats

    DEFF Research Database (Denmark)

    Müller, Anne Kirstine; Farombi, E.O.; Møller, P.

    2004-01-01

    , in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue(R) rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks...

  3. DAF-16/FOXO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage.

    Science.gov (United States)

    Mueller, Michael M; Castells-Roca, Laia; Babu, Vipin; Ermolaeva, Maria A; Müller, Roman-Ulrich; Frommolt, Peter; Williams, Ashley B; Greiss, Sebastian; Schneider, Jennifer I; Benzing, Thomas; Schermer, Bernhard; Schumacher, Björn

    2014-12-01

    Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature ageing. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with ageing. Here we show that the FOXO transcription factor DAF-16 is activated in response to DNA damage during development, whereas the DNA damage responsiveness of DAF-16 declines with ageing. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA-damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16-mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists.

  4. Studies on DNA Damage Response in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Han, Wenyuan

    global reactions known as DNA damage response (DDR). In Bacteria and Eukaryotes, the global reactions include a series of transcription regulations and protein post-translation modifications, which can activate DNA repair machineries, suppress cell division and delay DNA replication, and induce......) and methyl methanesulfonate (MMS), and hydroxyurea (HU) that may not introduce DNA lesions directly. Comparison of the effects of the three drugs on S. islandicus cells showed that NQO and MMS led to DNA-less cell formation, while HU did not. In addition, the DNA-less cells were featured with increased side...... scattered light, damaged cell membrane and electron-dense area. During NQO and MMS treatment, degradation of chromatin proteins was coincided with DNA-less cell formation, suggesting their roles in protecting genomic DNA from massive degradation. Further, HU inhibited NQO-induced DSB formation and DNA...

  5. Effect of ionizing radiation in sensory ganglion neurons: organization and dynamics of nuclear compartments of DNA damage/repair and their relationship with transcription and cell cycle.

    Science.gov (United States)

    Casafont, Iñigo; Palanca, Ana; Lafarga, Vanesa; Berciano, Maria T; Lafarga, Miguel

    2011-10-01

    Neurons are very sensitive to DNA damage induced by endogenous and exogenous genotoxic agents, as defective DNA repair can lead to neurodevelopmental disorders, brain tumors and neurodegenerative diseases with severe clinical manifestations. Understanding the impact of DNA damage/repair mechanisms on the nuclear organization, particularly on the regulation of transcription and cell cycle, is essential to know the pathophysiology of defective DNA repair syndromes. In this work, we study the nuclear architecture and spatiotemporal organization of chromatin compartments involved in the DNA damage response (DDR) in rat sensory ganglion neurons exposed to X-ray irradiation (IR). We demonstrate that the neuronal DDR involves the formation of two categories of DNA-damage processing chromatin compartments: transient, disappearing within the 1 day post-IR, and persistent, where unrepaired DNA is accumulated. Both compartments concentrate components of the DDR pathway, including γH2AX, pATM and 53BP1. Furthermore, DNA damage does not induce neuronal apoptosis but triggers the G0-G1 cell cycle phase transition, which is mediated by the activation of the ATM-p53 pathway and increased protein levels of p21 and cyclin D1. Moreover, the run on transcription assay reveals a severe inhibition of transcription at 0.5 h post-IR, followed by its rapid recovery over the 1 day post-IR in parallel with the progression of DNA repair. Therefore, the response of healthy neurons to DNA damage involves a transcription- and cell cycle-dependent but apoptosis-independent process. Furthermore, we propose that the segregation of unrepaired DNA in a few persistent chromatin compartments preserves genomic stability of undamaged DNA and the global transcription rate in neurons.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A 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

  7. Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells.

    Directory of Open Access Journals (Sweden)

    Luca A Petruccelli

    Full Text Available Histone deacetylase inhibitors (HDACi are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

  8. Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

    Science.gov (United States)

    Pettersson, Filippa; Retrouvey, Hélène; Skoulikas, Sophia; Miller, Wilson H.

    2011-01-01

    Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents. PMID:21695163

  9. Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair

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