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Sample records for dna strand damage

  1. What is DNA damage? Risk of double-strand break and its individual variation

    International Nuclear Information System (INIS)

    Hanaoka, Fumio

    2011-01-01

    The author discusses about the title subject in an aspect of possible spreading of Fukushima radioactive substances mainly in eastern north area of Japan where carcinogenic incidence may be increased as the ionizing radiation injures the gene (DNA). At first, explained is that cancer is a disease of genes with infinitive proliferation of cells, there are systems to prevent it by repairing the damaged DNA and by other mechanisms like exclusion of cells damaged too much or killing cancer cells with immunity, and individual difference of the repairing capability exists. DNA is always damaged even under ordinary living conditions by sunlight UV ray, cosmic radiation and chemicals externally and by active oxygen species and thermal water movement internally. Concomitantly, DNA damaged by many mechanisms like deletion, dimmer formation, chemical modification of bases, single and double strand breaks is always repaired by concerned enzymes. Double-strand damage by high-energy radiation like gamma ray is quite risky because its repair sometimes accompanies error as concerned enzymes are from more multiple genes. There are many syndromes derived from gene deficit of those repairing enzymes. The diseases concerned with repair of the double-strand damage teach that fetus and infant are more sensitive to radiation than adult as their young body cells are more actively synthesizing DNA, during which, if DNA is injured by radiation, risk of repairing error is higher as the double strand break more frequently occurs. It cannot be simply said that a certain radiation dose limit is generally permissible. There is an individual difference of radiation sensitivity and a possible method to find out an individual weak to radiation is the lymphocyte screening in vitro using anticancer bleomycin which breaks the double strand. (T.T.)

  2. Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.

    Science.gov (United States)

    Reis, António M C; Mills, Wilbur K; Ramachandran, Ilangovan; Friedberg, Errol C; Thompson, David; Queimado, Lurdes

    2012-01-01

    Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

  3. 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

  4. 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

    ; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage...... 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...

  5. Compound Poisson Processes and Clustered Damage of Radiation Induced DNA Double Strand Breaks

    International Nuclear Information System (INIS)

    Gudowska-Nowak, E.; Ritter, S.; Taucher-Scholz, G.; Kraft, G.

    2000-01-01

    Recent experimental data have demonstrated that DNA damage induced by densely ionizing radiation in mammalian cells is distributed along the DNA molecule in the form of clusters. The principal constituent of DNA damage are double-strand breaks (DSB) which are formed when the breaks occur in both DNA strands and are directly opposite or separated by only a few base pairs. DSBs are believed to be most important lesions produced in chromosomes by radiation; interaction between DSBs can lead to cell killing, mutation or carcinogenesis. The paper discusses a model of clustered DSB formation viewed in terms of compound Poisson process along with the predictive essay of the formalism in application to experimental data. (author)

  6. Detection of DNA damage in cells exposed to ionizing radiation by use of antisingle-stranded-DNA monoclonal antibody

    International Nuclear Information System (INIS)

    Schans, G.P. van der; Loon, A.A.W.M. van; Groenendijk, R.H.; Baan, R.A.

    1989-03-01

    An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody, D1B, was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the partial umwinding of cellular DNA under alkaline conditions, a time-dependent process. Single-strand and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding under controlled conditions is a measure, therefore, of the amount of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single- from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gu of ionizing radiation and needs only small amounts of cells. The usefulness of the technique was demonstrated in a study on the induction of damage and its repair in unlabelled cultured Chinese hamster cells and in DNA-containing cells of human blood, both after exposure to 60 Co-γ-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related degree of unwinding was observed and repair could be observed up to 60 min after irradiation. (author). 19 refs.; 3 figs.; 1 tab

  7. Assessment of evidence for nanosized titanium dioxide-generated DNA strand breaks and oxidatively damaged DNA in cells and animal models

    DEFF Research Database (Denmark)

    Møller, Peter; Jensen, Ditte Marie; Wils, Regitze Sølling

    2017-01-01

    Nanosized titanium dioxide (TiO2) has been investigated in numerous studies on genotoxicity, including comet assay endpoints and oxidatively damaged DNA in cell cultures and animal models. The results have been surprisingly mixed, which might be attributed to physico-chemical differences...... culture studies also demonstrate increased levels of oxidatively damaged DNA after exposure to TiO2. There are relatively few studies on animal models where DNA strand breaks and oxidatively damaged DNA have been tested with reliable methods. Collectively, this review shows that exposure to nanosized TiO2...... of the tested TiO2. In the present review, we assess the role of certain methodological issues and publication bias. The analysis shows that studies on DNA strand breaks without proper assay controls or very low intra-group variation tend to show statistically significant effects. Levels of oxidatively damaged...

  8. Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.

    Directory of Open Access Journals (Sweden)

    Kin Chan

    Full Text Available Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA is more prone to damage than double-strand DNA (dsDNA, due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5' to 3' enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3' ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2'-deoxyuracil intermediate in DNA that was resistant to excision by uracil-DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions

  9. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

  10. Delayed repair of DNA single-strand breaks does not increase cytogenetic damage

    International Nuclear Information System (INIS)

    Morgan, W.F.; Djordjevic, M.C.; Jostes, R.F.; Pantelias, G.E.

    1985-01-01

    DNA damage and cytogenetic effects of ionizing radiation were investigated in Chinese hamster ovary (CHO) cells and unstimulated human peripheral blood lymphocytes. DNA damage and repair were analysed by alkaline elution under conditions that predominantly measured DNA single-strand breaks (ssb). X-radiation (2.5 Gy) induced ssb in both CHO cells and unstimulated lymphocytes, and the breaks were repaired within 30 and 90 min, respectively. This rapid repair was delayed by the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB). The cytogenetic effects of the 3AB-induced delay in DNA repair were examined by analysing sister chromatid exchange (SCE) frequency in CHO cells and fragmentation of prematurely condensed chromosomes (PCC) in unstimulated human lymphocytes after 2.5 Gy of X-rays. Although 3AB delayed the rejoining of DNA ssb, this delay did not result in increased cytogenetic damage manifested as either SCE or fragmentation of PCC. These results indicate that the rapidly rejoining DNA ssb are not important in the production of chromosome damage. (author)

  11. Comparison of DNA strand-break simulated with different DNA models

    International Nuclear Information System (INIS)

    Xie, Wenzhang; Li, Junli; Qiu, Rui; Yan, Congchong; Zeng, Zhi; Li, Chunyan

    2013-01-01

    Full text of the publication follows. In Monte Carlo simulation of DNA damage, the geometric model of DNA is of great importance. To study the influence of DNA model on the simulation of DNA damage, three DNA models were created in this paper. They were a volume model and two atomic models with different parameters. Direct DNA strand-break induced by low-energy electrons were simulated respectively with the three models. The results show that most of the energy depositions in the DNA segments do not lead to strand-breaks. The simple single strand-break (SSB) tends to be the predominant damage type, and the contribution of complex double strand-break (DSB) to the total DSB cannot be neglected. Among the yields of all the three DNA target models applied here, the yields of the volume model are the highest, the yields of the atomic model with double van der Waals radii (r) take the second place, whereas the yields of the atomic model with single r come last. On average, the ratios of SSB yields are approximately equivalent to the corresponding ratios of the models' volume. However, there seems to be no clear relationship between the DSB yields and the models' volume. (authors)

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

    Science.gov (United States)

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

    2016-05-04

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

  13. Strand breaks and lethal damage in plasmid DNA subjected to 60CO-γirradiation

    International Nuclear Information System (INIS)

    Klimczak, U.

    1992-01-01

    Experiments with calf thymus DNA subjected to extracellular irradiation yield information on the role of direct and indirect effects in single-strand breakage, if this is evaluated with reference to the scavenger activity in respect of OH radicals. The role of the two processes in the occurrence of double-stand breaks and further damage leading to cell decay has so far remained largely obscure. It was the aim of the study described here to contribute to research in this field by performing in vitro experiments on biologically active DNA. For this purpose, DNA from pBR322 plasmids was irradiated in the presence of OH-radical scavengers. The number of single-strand and double-strand breaks was determined on the basis of the system's ability to eliminate OH radicals. In order to asses the influence of irradiation processes on the biological activity of DNA, investigations were carried out in E. coli for transformations caused by irradiated plasmid DNA. The results were interpreted in the light of theories about inhomogenous reaction kinetics put forward by Mark et al. (1989). It was finally discussed, which of the gamma-irradiation injuries occurring in DNA was to be held responsible for the inactivation of plasmid DNA and which enzymatic processes were additionally at work here. (orig./MG) [de

  14. Genome-wide mapping of DNA strand breaks.

    Directory of Open Access Journals (Sweden)

    Frédéric Leduc

    Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

  15. DNA strand breakage by 125I-decay in oligoDNA

    International Nuclear Information System (INIS)

    Lobachevsky, P.; Martin, R.F.

    1996-01-01

    Full text: A double-stranded oligodeoxynucleotide containing 125 I-dC in a defined location, with 5'- or 3'- 32 P-end-labelling of either strand, was used to investigate DNA strand breakage resulting from 125 I decay. Samples of the 32 P-end-labelled and 125 I-dC containing oligoDNA were incubated in 20 mM phosphate buffer (PB), or PB + 2 M dimethylsulphoxide (DMSO) at 4 deg during 18-20 days. The 32 P-end-labelled DNA fragments produced by 125 I decays were separated on denaturing polyacrylamide gels, and the 3P activity in each fragment was determined by scintillation counting after elution from the gel. The fragment size distribution was then converted to a distribution of single stranded break probabilities at each nucleotide position. The results indicate that each 125 I decay event produces at least one break in the 125 I-dC containing strand, and causes breakage of the opposite strand in 75-80% of events. Thus, the double stranded break is produced by 125 I decay with probability ∼0.8. Most of single stranded breaks (around 90%) occurred within 5-6 nucleotides of the 125 I-dC, however DNA breaks were detected up to 18-20 nucleotides from the decay site. The average numbers of single stranded breaks per decay are 3.7 (PB) and 3.3 (PB+DMSO) in 125 I-dC containing strand, and 1.5 (PB) and 1.3 (PB+DMSO) in the opposite strand. Deconvolution of strand break probabilities as a function of separation from the 125 I, in terms of both distance (to target deoxyribosyl carbon atoms, in B-DNA) and nucleotide number, show that the latter is an important parameter for the shorter-range damage. This could indicate a role for attenuation/dissipation of damage through the stacked bases. In summary, the results represent a much more extensive set of data than available from earlier experiments on DNA breakage from l25 I-decay, and may provide new mechanistic insights

  16. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  18. DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

    Science.gov (United States)

    Park, Sojin; Choi, Seoyun; Ahn, Byungchan

    2016-01-01

    DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

  19. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

    Science.gov (United States)

    Maréchal, Alexandre; Zou, Lee

    2015-01-01

    The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

  20. 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

  1. DN2 Thymocytes Activate a Specific Robust DNA Damage Response to Ionizing Radiation-Induced DNA Double-Strand Breaks

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    Irene Calvo-Asensio

    2018-06-01

    Full Text Available For successful bone marrow transplantation (BMT, a preconditioning regime involving chemo and radiotherapy is used that results in DNA damage to both hematopoietic and stromal elements. Following radiation exposure, it is well recognized that a single wave of host-derived thymocytes reconstitutes the irradiated thymus, with donor-derived thymocytes appearing about 7 days post BMT. Our previous studies have demonstrated that, in the presence of donor hematopoietic cells lacking T lineage potential, these host-derived thymocytes are able to generate a polyclonal cohort of functionally mature peripheral T cells numerically comprising ~25% of the peripheral T cell pool of euthymic mice. Importantly, we demonstrated that radioresistant CD44+ CD25+ CD117+ DN2 progenitors were responsible for this thymic auto-reconstitution. Until recently, the mechanisms underlying the radioresistance of DN2 progenitors were unknown. Herein, we have used the in vitro “Plastic Thymus” culture system to perform a detailed investigation of the mechanisms responsible for the high radioresistance of DN2 cells compared with radiosensitive hematopoietic stem cells. Our results indicate that several aspects of DN2 biology, such as (i rapid DNA damage response (DDR activation in response to ionizing radiation-induced DNA damage, (ii efficient repair of DNA double-strand breaks, and (iii induction of a protective G1/S checkpoint contribute to promoting DN2 cell survival post-irradiation. We have previously shown that hypoxia increases the radioresistance of bone marrow stromal cells in vitro, at least in part by enhancing their DNA double-strand break (DNA DSB repair capacity. Since the thymus is also a hypoxic environment, we investigated the potential effects of hypoxia on the DDR of DN2 thymocytes. Finally, we demonstrate for the first time that de novo DN2 thymocytes are able to rapidly repair DNA DSBs following thymic irradiation in vivo.

  2. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus leukocytes measured with the Comet and DNA diffusion assays

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    Adriana Díaz

    2009-01-01

    Full Text Available The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1 to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2 to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3 to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29% of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

  3. 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.

  4. Modeling the yield of double-strand breaks due to formation of multiply damaged sites in irradiated plasmid DNA

    International Nuclear Information System (INIS)

    Xapsos, M.A.; Pogozelski, W.K.

    1996-01-01

    Although double-strand breaks have long been recognized as an important type of DNa lesion, it is well established that this broad class of damage does not correlate well with indicators of the effectiveness of radiation as the cellular level. Assays of double-strand breaks do not distinguish the degree of complexity or clustering of singly damaged sites produced in a single energy deposition event, which is currently hypothesized to be key to understanding cellular end points. As a step toward this understanding, double-strand breaks that are formed proportionally to dose in plasmid DNA are analyzed from the mechanistic aspect to evaluate the yield that arises from multiply damaged sites as hypothesized by Ward (Prog. Nucleic Acid Res. Mol. Biol. 35, 95-125, 1988) and Goodhead (Int. J. Radiat. Biol. 65, 7-17, 1994) as opposed to the yield that arises form single hydroxyl radicals as hypothesized by Siddiqi and Bothe (Radiat. Res. 112, 449-463, 1987). For low-LET radiation such as γ rays, the importance of multiply damaged sites is shown to increase with the solution's hydroxyl radical scavenging capacity. For moderately high-LET radiation such as 100 keV/μm helium ions, a much different behavior is observed. In this case, a large fraction of double-strand breaks are formed as a result of multiply damaged sties over a broad range of scavenging conditions. Results also indicate that the RBE for common cellular end points correlates more closely with the RBE for common cellular end points correlates more closely with the RBE for multiply damaged sites than with the RBE for total double-strand breaks over a range of LET up to at least 100 keV/μm. 22 refs., 3 figs., 2 tabs

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

    Science.gov (United States)

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

    2013-10-01

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

  6. DNA double-strand breaks & poptosis in the testis

    NARCIS (Netherlands)

    Hamer, Geert

    2003-01-01

    During spermatogenesis, DNA damage is a naturally occurring event. At a certain stage, during the first meiotic prophase, DNA breaks are endogenously induced and even required for meiotic recombination. We studied these DNA breaks but also used ionizing radiation (IR) to induce DNA double-strand

  7. Using DNA origami nanostructures to determine absolute cross sections for UV photon-induced DNA strand breakage.

    Science.gov (United States)

    Vogel, Stefanie; Rackwitz, Jenny; Schürman, Robin; Prinz, Julia; Milosavljević, Aleksandar R; Réfrégiers, Matthieu; Giuliani, Alexandre; Bald, Ilko

    2015-11-19

    We have characterized ultraviolet (UV) photon-induced DNA strand break processes by determination of absolute cross sections for photoabsorption and for sequence-specific DNA single strand breakage induced by photons in an energy range from 6.50 to 8.94 eV. These represent the lowest-energy photons able to induce DNA strand breaks. Oligonucleotide targets are immobilized on a UV transparent substrate in controlled quantities through attachment to DNA origami templates. Photon-induced dissociation of single DNA strands is visualized and quantified using atomic force microscopy. The obtained quantum yields for strand breakage vary between 0.06 and 0.5, indicating highly efficient DNA strand breakage by UV photons, which is clearly dependent on the photon energy. Above the ionization threshold strand breakage becomes clearly the dominant form of DNA radiation damage, which is then also dependent on the nucleotide sequence.

  8. 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.

  9. Radiation and non-radiation damage to DNA. Onset of molecular instability and carcinogenesis. Theoretical explorations on DNA damage and repair

    International Nuclear Information System (INIS)

    Pinak, Miroslay; Bunta, J.K.

    2006-01-01

    The current work is focused on results of molecular dynamics simulations performed on two DNA damages: 8-oxoguanine as the most significant oxidative damage leading to transversion mutation cytosine-guanine→adenine-thymine', which is common mutation found in human cancer cells; and on the DNA strand break, the type of damage that is considered to be one of the most significant damage leading to genetic instability that may result in enhanced cell proliferation or carcinogenesis. Except the structural changes induced by these two lesions the role and importance of electrostatic energy in recognition process in which a respective repair enzyme recognizes damaged DNA site is also described. Among the significant results can be included the fact, that most of the damages on DNA alternate locally electronic state by modifying chemical and electron orbital configuration. This modified configuration may be represented outside DNA molecule as an enhanced electrostatic interaction with surrounding environment, that may signal the presence of the damaged site toward the repair enzyme. Work on the DNA strand break shows that open valences at broken strand ends are quickly filled by the electrons generated during radiolysis. Results of simulation indicate a local instability of hydrogen bonds between complementary bases. (author)

  10. Repair response for DNA double-strand damage through ubiquitylation of chromatin

    International Nuclear Information System (INIS)

    Nakada, Shinichiro

    2011-01-01

    The chromatin modulation (remodeling) via lysine63 (K63)-linked ubiquitin (U) has been found important in the repair response for DNA double-strand damage, and the sequential signaling events at the damage site are explained. As the first step of the repair, MRN (MRE11, RAD50 and nibrin) complex recognizes the damage site and binds to it followed by many linked reactions by recruited and activated enzymes of various protein kinases and phosphatases, which resulting in the enhanced early signaling. As well, gamma-H2AX (phosphorylated histone H2AX) is yielded by the process, to which phosphorylated MDC1 (mediator of DNA-damage checkpoint 1) binds to produce their complex. Then further binding of RNF8-HERC2-UBC13 (ring finger protein 8, hect domain and RCC1 (CHC1)-like domain, and U conjugating enzyme E2N, respectively) occurs for starting the cumulative ubiquitylation of H2AX via K63 as the middle phase response. Signaling in the late phase occurs on the U chain formed at the damage site by binding of RAP (receptor-associated protein) 80 and other recruited 5 proteins like BRCA1 (breast cancer 1, early onset) to repair DNA by the homologous recombination after 53BP1 (tumor protein p53 binding protein) binding followed by methylation of histone H4. In a case of human compound heterozygous RNF168 defect, RIDDLE syndrome (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties), cells have no and slight abnormality of G2/M and intra-S checkpoint, respectively. Another defecting case with homozygous nonsense mutation has high radiosensitivity, intra-S checkpoint abnormality and others. Abnormality of immuno-globulins observed in both cases is similar to that in the RNF8-knockout mouse. Many tasks in chromatin ubiquitylation in the repair are still remained to be solved for protection and treatment of related diseases. (T.T.)

  11. 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

  12. Biologically important radiation damage in DNA

    International Nuclear Information System (INIS)

    Ward, J.F.

    1994-01-01

    Most DNA damage by the hydroxyl radical is confined to the bases, and this base damage represents an important component of locally multiply demanded sites (LMOS). The yields of the major damaged bases have been determined by gas chromatography mass spectrometry. For our propose, it was necessary to convert a known fraction of these damaged bases to strand breaks and then assay these labile sites as the increase in strand break yield over the normally observed level. Three potential agents by which this strategy of conversion of base damage to strand break could be implemented were identified in the original application: 1, Sl nuclease; 2, piperidine; and 3, base damage specific enzymes

  13. 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.

  14. 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.

  15. Un-repairable DNA damage in cell due to irradiation

    International Nuclear Information System (INIS)

    Yoshii, Giichi

    1992-01-01

    Radiation-induced cell reproductive deactivation is caused by damage to DNA. In a cell, cellular DNA radical reacts with diffusion controlled rate and generates DNA peroxide radical. The chemical repair of DNA radical with hydrogen donation by thiol competes with the reaction of oxygen with same radicals in the DNA molecules. From the point reaction rates, the prolongation of radical life time is not as great as expected from the reduction in the glutathione content of the cell. This indicates that further reducting compounds (protein bound thiol) are present in the cell. The residual radicals are altered to strand breaks, base damages and so on. The effective lesions for a number of endpoints is un-repaired double strand break, which has been discovered in a cluster. This event gives risk to high LET radiation or to a track end of X-rays. For X- or electron irradiations the strand breaks are frequently induced by the interactions between sublesions on two strands in DNA. A single strand break followed by radical action may be unstable excited state, because of remaining sugar radical action and of having negative charged phosphates, in which strands breaks will be rejoined in a short time to stable state. On the same time, a break in the double helix will be immediately produced if two breaks are on either or approximately opposite locations. The formation of a double strand break in the helix depends on the ion strength of the cell. The potassium ions are largely released from polyanionic strand during irradiation, which results in the induction of denatured region. Double strand break with the denatured region seems to be un-repairable DNA damage. (author)

  16. Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

    Science.gov (United States)

    Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

    2017-10-19

    Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Effects of hyperthermia on repair of radiation-induced DNA strand breaks

    International Nuclear Information System (INIS)

    Mills, M.D.; Meyn, R.E.

    1981-01-01

    Previous reports have suggested a relationship between the heat-induced changes in nucleoprotein and the hyperthermic enhancement of radiation sensitivity. In an effort to further understand these relationships, we measured the level of initial DNA strand break damage and the DNA strand break rejoining kinetics in Chinese hamster ovary cells following combined hyperthermia and ionizing radiation treatments. The amount of protein associated with DNA measured as the ratio of [ 3 H)leucine to [ 14 C]thymidine was also compared in chromatin isolated from both heated and unheated cells. The results of these experiments show that the initial level of radiation-induced DNA strand breaks is significantly enhanced by a prior hyperthermia treatment of 43 0 C for 30 min. Treatments at higher temperatures and longer treatments at the same temperature magnified this effect. Hyperthermia was also shown to cause a substantial inhibition of the DNA strand break rejoining after irradiation. Both the initial level of DNA damage and the rejoining kinetics recovered to normal levels with incubation at 37 0 C between the hyperthermia and radiation treatments. Recovery of these parameters coincided with the return of the amount of protein associated with DNA to normal values, further suggesting a relationship between the changes in nucleoprotein and the hyperthermic enhancement of radiation sensivivity

  18. 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.

  19. Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

    Science.gov (United States)

    Caldecott, K W

    2001-05-01

    The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

  20. 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

  1. A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

    Science.gov (United States)

    Calderwood, Stuart K

    2016-05-26

    Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

  2. Meta-analysis of DNA double-strand break response kinetics

    NARCIS (Netherlands)

    Kochan, Jakub A.; Desclos, Emilie C. B.; Bosch, Ruben; Meister, Luna; Vriend, Lianne E. M.; Attikum, Haico V.; Krawczyk, Przemek M.

    2017-01-01

    Most proteins involved in the DNA double-strand break response (DSBR) accumulate at the damage sites, where they perform functions related to damage signaling, chromatin remodeling and repair. Over the last two decades, studying the accumulation of many DSBR proteins provided information about their

  3. 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

  4. Strand breaks in plasmid DNA following positional changes of Auger-electron-emitting radionuclides

    International Nuclear Information System (INIS)

    Adelstein, S.J.; Kassis, A.I.

    1996-01-01

    The purpose of our studies is to elucidate the kinetics of DNA strand breaks caused by low-energy Auger electron emitters in close proximity to DNA. Previously we have studied the DNA break yields in plasmids after the decay of indium-111 bound to DNA or free in solution. In this work, we compare the DNA break yields in supercoiled DNA of iodine-125 decaying close to DNA following DNA intercalation, minor-groove binding, or surface binding, and at a distance form DNA. Supercoiled DNA, stored at 4 C to accumulate radiation dose from the decay of 125 I, was then resolved by gel electrophoresis into supercoiled, nicked circular, and linear forms, representing undamaged DNA, single-strand breaks, and double-strand breaks respectively. DNA-intercalated or groove-bound 125 I is more effective than surface-bound radionuclide or 125 I free in solution. The hydroxyl radical scavenger DMSO protects against damage by 125 I free in solution but has minimal effect on damage by groove-bound 125 I. (orig.)

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

    Science.gov (United States)

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

    2016-01-01

    A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

  6. 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.

  7. 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

  8. 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.

  9. Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1994-01-01

    Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

  10. 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.)

  11. Repair of Clustered Damage and DNA Polymerase Iota.

    Science.gov (United States)

    Belousova, E A; Lavrik, O I

    2015-08-01

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

  12. The Human L1 Element Causes DNA Double-Strand Breaks in Breast Cancer

    Science.gov (United States)

    2006-08-01

    cancer is complex. However, defects in DNA repair genes in the double-strand break repair pathway are cancer predisposing. My lab has characterized...a new potentially important source of double-strand breaks (DSBs) in human cells and are interested in characterizing which DNA repair genes act on...this particular source of DNA damage. Selfish DNA accounts for 45% of the human genome. We have recently demonstrated that one particular selfish

  13. 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)

  14. The yield, processing, and biological consequences of clustered DNA damage induced by ionizing radiation

    International Nuclear Information System (INIS)

    Shikazono, Naoya; Noguchi, Miho; Fujii, Kentaro; Urushibara, Ayumi; Yokoya, Akinari

    2009-01-01

    After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. The DNA lesions include single strand breaks (SSB), base lesions, sugar damage, and apurinic/apyrimidinic sites (AP sites). Clustered DNA damage, which is defined as two or more of such lesions within one to two helical turns of DNA induced by a single radiation track, is considered to be a unique feature of ionizing radiation. A double strand break (DSB) is a type of clustered DNA damage, in which single strand breaks are formed on opposite strands in close proximity. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints, such as cell death, loss of genetic material, chromosome aberration. Although non-DSB clustered DNA damage has received less attention, there is growing evidence of its biological significance. This review focuses on the current understanding of (1) the yield of non-DSB clustered damage induced by ionizing radiation (2) the processing, and (3) biological consequences of non-DSB clustered DNA damage. (author)

  15. Effects of motexafin gadolinium on DNA damage and X-ray-induced DNA damage repair, as assessed by the Comet assay

    International Nuclear Information System (INIS)

    Donnelly, Erling T.; Liu Yanfeng; Paul, Tracy K.; Rockwell, Sara

    2005-01-01

    Purpose: To investigate the effects of motexafin gadolinium (MGd) on the levels of reactive oxygen species (ROS), glutathione (GSH), and DNA damage in EMT6 mouse mammary carcinoma cells. The ability of MGd to alter radiosensitivity and to inhibit DNA damage repair after X-ray irradiation was also evaluated. Methods and Materials: Reactive oxygen species and GSH levels were assessed by 2,7-dichlorofluorescein fluorescence flow cytometry and the Tietze method, respectively. Cellular radiosensitivity was assessed by clonogenic assays. Deoxyribonucleic acid damage and DNA damage repair were assessed in plateau-phase EMT6 cells by the Comet assay and clonogenic assays. Results: Cells treated with 100 μmol/L MGd plus equimolar ascorbic acid (AA) had significantly increased levels of ROS and a 58.9% ± 3.4% decrease in GSH levels, relative to controls. Motexafin gadolinium plus AA treatment increased the hypoxic, but not the aerobic, radiosensitivity of EMT6 cells. There were increased levels of single-strand breaks in cells treated with 100 μmol/L MGd plus equimolar AA, as evidenced by changes in the alkaline tail moment (MGd + AA, 6 h: 14.7 ± 1.8; control: 2.8 ± 0.9). The level of single-strand breaks was dependent on the length of treatment. Motexafin gadolinium plus AA did not increase double-strand breaks. The repair of single-strand breaks at 2 h, but not at 4 h and 6 h, after irradiation was altered significantly in cells treated with MGd plus AA (MGd + AA, 2 h: 15.8 ± 3.4; control: 5.8 ± 0.6). Motexafin gadolinium did not alter the repair of double-strand breaks at any time after irradiation with 10 Gy. Conclusions: Motexafin gadolinium plus AA generated ROS, which in turn altered GSH homeostasis and induced DNA strand breaks. The MGd plus AA-mediated alteration of GSH levels increased the hypoxic, but not aerobic, radiosensitivity of EMT6 cells. Motexafin gadolinium altered the kinetics of single-strand break repair soon after irradiation but did not

  16. Detection of DNA strand breaks in mammalian cells using the radioresistant bacterium PprA protein

    International Nuclear Information System (INIS)

    Satoh, Katsuya; Wada, Seiichi; Narumi, Issay; Kikuchi, Masahiro; Funayama, Tomoo; Kobayashi, Yasuhiko

    2003-01-01

    We have previously found that the PprA protein from Deinococcus radiodurans possesses ability to recognize DNA carrying strand breaks. In the present study, we attempted to visualize radiation-induced DNA strand breaks with PprA protein using immunofluorescence technique to elucidate the DNA damage response mechanism in mammalian cultured cells. As a result, colocalization of Cy2 and DAPI fluorescent signals was observed. This observation suggests that DNA strand breaks in the nucleus of CHO-K1 cells were effectively detected using the PprA protein. The amount of DNA strand breaks (integrated density of Cy2 fluorescent signals) was increased with the increase in the radiation dose. (author)

  17. Electrolytic reduction of nitroheterocyclic drugs leads to biologically important damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, M.V.M.; Pluijmackers-Westmijze, E.J.; Loman, H.

    1985-01-01

    The effects of electrolytic reduction of nitroimidazole drugs on biologically active DNA was studied. The results show that reduction of the drugs in the presence of DNA affects inactivation for both double-stranded (RF) and single-stranded phiX174 DNA. However, stable reduction products did not make a significant contribution to the lethal damage in DNA. This suggests that probably a short-lived intermediate of reduction of nitro-compounds is responsible for damage to DNA. (author)

  18. Simulation of 125I-induced DNA strand breaks in a CAP-DNA complex

    International Nuclear Information System (INIS)

    Li, W.; Friedland, W.; Jacob, P.

    2000-01-01

    DNA strand breakage induced by decay of 125 I incorporated into the pyrimidine of a small piece of DNA with a specific base pair sequence has been investigated theoretically and experimentally (Lobachevsky and Martin 2000a, 2000b; Nikjoo et al., 1996; Pomplun and Terrissol, 1994; Charlton and Humm, 1988). Recently an attempt was made to analyse the DNA kinks in a CAP-DNA complex with 125 I induced DNA strand breakage (Karamychev et al., 1999). This method could be used as a so called radioprobing for such DNa distortions like other chemical and biological assays, provided that it has been tested and confirmed in a corresponding theoretical simulation. In the measurement, the distribution of the first breaks on the DNA strands starting from their labeled end can be determined. Based on such first breakage distributions, the simulation calculation could then be used to derive information on the structure of a given DNA-protein complex. The biophysical model PARTRAC has been applied successfully in simulating DNA damage induced by irradiation (Friedland et al., 1998; 1999). In the present study PARTRAC is adapted to a DNA-protein complex in which a specific sequence of 30 base pairs of DNA is connected with the catabolite gene activator protein (CAP). This report presents the first step of the analysis in which the CAP-DNA model used in NIH is overlaid with electron track structures in liquid water and the strand breaks due to direct ionization and due to radical attack are simulated. The second step will be to take into account the neutralization of the heavily charged tellurium and the protective effect of the CAP protein against radical attack. (orig.)

  19. DNA double strand break repair is enhanced by P53 following induction by DNA damage and is dependent on the C-terminal domain of P53

    International Nuclear Information System (INIS)

    Wei Tang; Powell, Simon N.

    1996-01-01

    Purpose: The tumor suppressor gene p53 can mediate cell cycle arrest or apoptosis in response to DNA damage. Accumulating evidence suggests that it may also directly or indirectly influence the DNA repair machinery. In the present study, we investigated whether p53, induced by DNA damage, could enhance the rejoining of double-strand DNA breaks. Materials and Methods: DNA double-strand breaks (dsb) were made by restriction enzyme digestion of a plasmid, between a promoter and a 'reporter' gene: luciferase (LUC) or chloramphenicol acetyl-transferase (CAT). Linear or circular plasmid DNA (LUC or CAT) was co-transfected with circular β-Gal plasmid (to normalize for uptake) into mouse embryonic fibroblasts genetically matched to be (+/+) or (-/-) for p53. Their ability to rejoin linearized plasmid was measured by the luciferase or CAT activity detected in rescued plasmids. The activity detected in cells transfected with linear plasmid was scored relative to the activity detected in cells transfected with circular plasmid. Results: Ionizing radiation (IR, 2 Gy) enhanced the dsb repair activity in wild type p53 cells; however, p53 null cells lose this effect, indicating that the enhancement of dsb repair was p53-dependent. REF cells with dominant-negative mutant p53 showed a similar induction compared with the parental REF cells with wild-type p53. This ala-143 mutant p53 prevents cell cycle arrest and transactivation of p21 WAF1/cip1) following IR, indicating that the p53-dependent enhancement of DNA repair is distinct from transactivation. Immortalized murine embryonic fibroblasts, 10(1)VasK1 cells, which express p53 cDNA encoding a temperature-sensitive mutant in the DNA sequence specific binding domain (ala135 to val135) with an alternatively spliced C-terminal domain (ASp53: amino-acids 360-381) and, 10(1)Val5 cells, which express the normal spliced p53 (NSp53) with the same temperature-sensitive mutant were compared. It was found that 10(1)VasK1 cells showed no DNA

  20. 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)

  1. Breaking DNA strands by extreme-ultraviolet laser pulses in vacuum

    Czech Academy of Sciences Publication Activity Database

    Nováková, Eva; Vyšín, Luděk; Burian, Tomáš; Juha, Libor; Davídková, Marie; Múčka, V.; Čuba, V.; Grisham, M. E.; Heinbuch, S.; Rocca, J.J.

    2015-01-01

    Roč. 91, č. 4 (2015), "042718-1"-"042718-8" ISSN 1539-3755 R&D Projects: GA ČR(CZ) GBP108/12/G108; GA ČR GA13-28721S Institutional support: RVO:68378271 ; RVO:61389005 Keywords : XUV * DNA damages * single- strand breaks (SSBs) * double- strand breaks (DSBs) Subject RIV: BO - Biophysics Impact factor: 2.288, year: 2014

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Science.gov (United States)

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

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

  4. Defective processing of methylated single-stranded DNA by E. coli alkB mutants

    Science.gov (United States)

    Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

    2000-01-01

    Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

  5. Radiation-induced DNA damage in halogenated pyrimidine incorporated cells and its correlation with radiosensitivity

    International Nuclear Information System (INIS)

    Watanabe, R.; Nikjoo, H.

    2003-01-01

    Cells with DNA containing 5-halogenated pyrimidines in place of thymidine show significant reductions of slope (Do) and shoulder (Dq) of their radiation survival curves. Similar radiosensitization has also been observed in the yield of DNA strand breaks. The purpose of this study is to obtain an insight into the mechanism of cell lethality by examining the relationship between the spectrum of DNA damage and the cell survival. In this study we estimated the enhancement of strand breaks due to incorporation of halogenated pyrimidine, the complexity of DNA damage and the probability of the initial DNA damage leading to cell inactivation. Monte Carlo track structure methods were used to model and simulate the induction of strand breakage by X-rays. The increase of DNA strand break was estimated by assuming the excess strand break was caused by the highly reactive uracil radicals at the halouracil substituted sites. The assumption of the enhancement mechanism of strand breaks was examined and verified by comparison with experimental data for induction of SSB and DSB. The calculated DNA damage spectrum shows the increase in complexity of strand breaks is due to incorporation of halogenated pyrimidines. The increase in the yield of DSB and cell lethality show similar trend at various degrees of halogenated pyrimidine substitution. We asked the question whether this agreement supports the hypothesis that DSB is responsible for cell lethality? The estimated number of lethal damage from the cell survival using a linear-quadratic model is much less than the initial yield of DSB. This work examines the correlation of cell lethality as a function of frequencies of complex form of double strand breaks

  6. Deficiency of Double-Strand DNA Break Repair Does Not Impair Mycobacterium tuberculosis Virulence in Multiple Animal Models of Infection

    OpenAIRE

    Heaton, Brook E.; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C.; Glickman, Michael S.

    2014-01-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA bre...

  7. Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template*

    OpenAIRE

    Yuan, Quan; McHenry, Charles S.

    2009-01-01

    In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of γ-complex to support the reaction in the absence of τ. However, if γ-complex is p...

  8. Protection of free-radical induced DNA strand breaks in vitro by flavonoids

    International Nuclear Information System (INIS)

    Fisher, L.; Anderson, R.F.

    1998-01-01

    Full text: We have used both plasmid and cosmid test systems to assay the effect of antioxidant flavonoids (AO) on DNA strand breakage in supercoiled closed circular DNA (DNA SC ) following the formation oxidative radical damage on DNA (DNA OXID + . ) in aqueous solution. Single strand breaks in DNA SC result in the formation of the relaxed circular form (DNA RC ) and double strand breaks give linear DNA (DNA L ). Dose response curves were constructed for the log of the loss of [DNA S C] against dose (0-600 Gy). The D 37 (dose for 37% unchanged DNA SC ) values determined in the presence of increasing amounts of flavonoids were compared as ratios to the D 37 control value to give dose modification factor (DMF). Irradiations were carried out under 'constant scavenging' conditions to separate out the effect of direct radical scavenging from the possible electron transfer reaction. Control irradiation experiments, were performed in aerated TRIS buffer, concentration 10 mM, which has a scavenging capacity, k s (defined as the summation of the rate constants for the reaction of OH radicals with all species in solution, multiplied by their concentrations) of 1.5 x 10 7 s -1 . The concentration of TRIS was reduced upon addition of AO to maintain k s at this level. Data will be presented for examples from all four major types of flavonoids (flavonols, isoflavones, flavones and flavon-3-ols) showing DMF values plateau at near 2.0 even at low concentrations (ca. 20 μM) of the flavonoids. Increased DNA strand breaks following post irradiation incubation with endo III protein was unaffected by having the flavonoids present at the time of irradiation. This result suggests that the protection afforded by the flavonoids is unlikely to be in repairing radical damage on pyrimidine bases that are precursors of DNA strand breaks. Overall these studies provide evidence for an additional mechanism of antioxidant activity

  9. GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

    2015-01-01

    Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

  10. Radiobiology of DNA strand breakage

    International Nuclear Information System (INIS)

    Johansen, I.

    1975-01-01

    The yield of single-strand breaks in lambda DNA within lysogenic host bacteria was measured after exposure to 4-MeV electrons (50 msec) and rapid transfer (45 msec) to alkaline detergent. In nitrogen anoxia the yield was 1.2 x 10 -12 DNA single-strand breaks per rad per dalton, and under full oxygenation the yield increased to 5 x 10 -12 breaks per rad per dalton. A search for the presence of fast repair mechanisms failed to demonstrate the presence of any mechanism for repair of strand breaks operating within a fraction of a second. Strand breaks produced in the presence of oxygen were repaired in 30--40 sec, while breaks produced under anoxia were rejoined even slower. A functional product from the polAl gene was needed for the rejoining of the broken molecules. Intermediate levels of DNA strand breakage seen at low concentrations of oxygen are dependent on the concentration of cellular sulfhydryl compounds, suggesting that in strand breakage oxygen and hydrogen donors compete for reactions with radiation-induced transients in the DNA. Intercomparisons of data on radiation-induced lethality of cells and single-strand breaks in episomal DNA allow the distinction between two classes of radiation-induced radicals, R 1 and R 2 , with different chemical properties; R 1 reacts readily with oxygen and N-oxyls under formation of potentially lethal products. The reactivity of oxygen in this reaction is 30--40 times higher than that of TMPN. R 2 reacts 16 times more readily than R 1 with oxygen under formation of single-strand breaks in the DNA. R 2 does not react with N-oxyls

  11. UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

    Science.gov (United States)

    Greinert, R.; Volkmer, B.; Henning, S.; Breitbart, E. W.; Greulich, K. O.; Cardoso, M. C.; Rapp, Alexander

    2012-01-01

    UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G1-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer. PMID:22941639

  12. Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells.

    LENUS (Irish Health Repository)

    Dodson, Helen

    2009-10-01

    The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.

  13. Stalled repair of lesions when present within a clustered DNA damage site

    International Nuclear Information System (INIS)

    Lomax, M.E.; Cunniffe, S.; O'Neill, P.

    2003-01-01

    Ionising radiation produces clustered DNA damages (two or more lesions within one or two helical turns of the DNA) which could challenge the repair mechanism(s) of the cell. Using purified base excision repair (BER) enzymes and synthetic oligonucleotides a number of recent studies have established the excision of a lesion within clustered damage sites is compromised. Evidence will be presented that the efficiency of repair of lesions within a clustered DNA damage site is reduced, relative to that of the isolated lesions, since the lifetime of both lesions is extended by up to four fold. Simple clustered damage sites, comprised of single-strand breaks, abasic sites and base damages, one or five bases 3' or 5' to each other, were synthesised in oligonucleotides and repair carried out in mammalian cell nuclear extracts. The rate of repair of the single-strand break/abasic site within these clustered damage sites is reduced, mainly due to inhibition of the DNA ligase. The mechanism of repair of the single-strand break/abasic site shows some asymmetry. Repair appears to be by the short-patch BER pathway when the lesions are 5' to each other. In contrast, when the lesions are 3' to each other repair appears to proceed along the long-patch BER pathway. The lesions within the cluster are processed sequentially, the single-strand break/abasic site being repaired before excision of 8-oxoG, limiting the formation of double-strand breaks to <2%. Stalled processing of clustered DNA damage extends the lifetime of the lesions to an extent that could have biological consequences, e.g. if the lesions are still present during transcription and/or at replication mutations could arise

  14. Strand breaks, base release and post-irradiation changes in DNA γ-irradiated in dilute O2-saturated aqueous solution

    International Nuclear Information System (INIS)

    Ward, J.F.; Kuo, I.

    1976-01-01

    Gamma irradiation of DNA in dilute O 2 -saturated aqueous solution releases free bases and damaged bases from the macromolecule. The yields of these products were measured after column chromatographic separation. For double stranded DNA the immediate yield of bases varies from G = 0.012 for cytosine to G = 0.033 for adenine. The yields of released bases increase with post-irradiation time (the majority of the increase occurs in the first 2 hrs.) to yields in the range of G = 0.07 +- 0.012. Yields of two released damaged thymine radiation products from γ-irradiated 3 H thymine labelled DNA also increased with post-irradiation time. Strand breaks were measured in γ-irradiated single stranded DNA the initial yield G = 0.02 was low but increased with time to G = 0.07. No direct correlation between strand-break production and release of low molecular weight products is possible. The findings are discussed in terms of damage to DNA in vivo and its enzymatic repair

  15. Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking.

    Science.gov (United States)

    Nakamura, Shigetaka; Hashimoto, Hirokazu; Kobayashi, Satoshi; Fujimoto, Kenzo

    2017-10-18

    DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole ( CNV K) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Radiation induced DNA double-strand breaks in radiology; Strahleninduzierte DNA-Doppelstrangbrueche in der Radiologie

    Energy Technology Data Exchange (ETDEWEB)

    Kuefner, M.A. [Dornbirn Hospital (Austria). Dept. of Radiology; Brand, M.; Engert, C.; Uder, M. [Erlangen University Hospital (Germany). Dept. of Radiology; Schwab, S.A. [Radiologis, Oberasbach (Germany)

    2015-10-15

    Shortly after the discovery of X-rays, their damaging effect on biological tissues was observed. The determination of radiation exposure in diagnostic and interventional radiology is usually based on physical measurements or mathematical algorithms with standardized dose simulations. γ-H2AX immunofluorescence microscopy is a reliable and sensitive method for the quantification of radiation induced DNA double-strand breaks (DSB) in blood lymphocytes. The detectable amount of these DNA damages correlates well with the dose received. However, the biological radiation damage depends not only on dose but also on other individual factors like radiation sensitivity and DNA repair capacity. Iodinated contrast agents can enhance the x-ray induced DNA damage level. After their induction DSB are quickly repaired. A protective effect of antioxidants has been postulated in experimental studies. This review explains the principle of the γ-H2AX technique and provides an overview on studies evaluating DSB in radiologic examinations.

  17. Monophosphate end groups produced in radiation induced strand breakage in DNA

    International Nuclear Information System (INIS)

    Kay, E.; Ward, J.F.

    1976-01-01

    A solution of DNA was gamma-irradiated and treated with monophosphatase for studies on the amount of inorganic phosphate released as a function of time. Studies were also conducted on: effect of alkali on yield of monophosphate end groups; induction of DNA strand breaks by treatment with DNAase; initial G values for monophosphate termini; and effect of alkali on radioinduced DNA damage

  18. Defective double-strand DNA break repair and chromosomal translocations by MYC overexpression.

    Science.gov (United States)

    Karlsson, Asa; Deb-Basu, Debabrita; Cherry, Athena; Turner, Stephanie; Ford, James; Felsher, Dean W

    2003-08-19

    DNA repair mechanisms are essential for the maintenance of genomic integrity. Disruption of gene products responsible for DNA repair can result in chromosomal damage. Improperly repaired chromosomal damage can result in the loss of chromosomes or the generation of chromosomal deletions or translocations, which can lead to tumorigenesis. The MYC protooncogene is a transcription factor whose overexpression is frequently associated with human neoplasia. MYC has not been previously implicated in a role in DNA repair. Here we report that the overexpression of MYC disrupts the repair of double-strand DNA breaks, resulting in a several-magnitude increase in chromosomal breaks and translocations. We found that MYC inhibited the repair of gamma irradiation DNA breaks in normal human cells and blocked the repair of a single double-strand break engineered to occur in an immortal cell line. By spectral karyotypic analysis, we found that MYC even within one cell division cycle resulted in a several-magnitude increase in the frequency of chromosomal breaks and translocations in normal human cells. Hence, MYC overexpression may be a previously undescribed example of a dominant mutator that may fuel tumorigenesis by inducing chromosomal damage.

  19. Why soft UV-A damages DNA: An optical micromanipulation study

    Science.gov (United States)

    Rapp, A.; Greulich, K. O.

    2013-09-01

    Optical micromanipulation studies have solved a puzzle on DNA damage and repair. Such knowledge is crucial for understanding cancer and ageing. So far it was not understood, why the soft UV component of sunlight, UV-A, causes the dangerous DNA double strand breaks. The energy of UV-A photons is below 4 eV per photon, too low to directly cleave the corresponding chemical bonds in DNA. This is occasionally used to claim that artificial sunbeds, which mainly use UV-A, would not impose a risk on health. UV-A is only sufficient for induction of single strand breaks. The essential new observation is that, when on the opposite strand there is another single strand break at a distance of up to 20 base pairs. These two breaks will be converted into a break of the whole double strand with all its known consequences for cancer and ageing. However, in natural sun the effect is counteracted. Simultaneous red light illumination reduces UV induced DNA damages to 1/3. Since sunlight has a red component, skin tanning with natural sun is not as risky as might appear at a first glance.

  20. ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses.

    Science.gov (United States)

    Berger, N Daniel; Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

    2017-10-05

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  1. 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.

  2. 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.

  3. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  4. GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Svetlana Kostyuk

    2015-01-01

    Full Text Available Background. Cell free DNA (cfDNA circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci. As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR, PCNA (FACS and antiapoptotic genes (BCL2 (RT-PCR and FACS, BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR. Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs. Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR, in the level of fatty acid binding protein FABP4 (FACS analysis and in the level of fat (Oil Red O. Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

  5. Binding to the minor groove of the double-strand, tau protein prevents DNA from damage by peroxidation.

    Science.gov (United States)

    Wei, Yan; Qu, Mei-Hua; Wang, Xing-Sheng; Chen, Lan; Wang, Dong-Liang; Liu, Ying; Hua, Qian; He, Rong-Qiao

    2008-07-02

    Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H(2)O(2)/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.

  6. Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

    Science.gov (United States)

    Yuan, Quan; McHenry, Charles S

    2009-11-13

    In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

  7. A model treating the DNA double-strand break repair inhibition by damage clustering

    International Nuclear Information System (INIS)

    Rosemann, M.; Abel, H.; Regel, K.

    1992-01-01

    A microdosimetric model for the interpretation of radiation induced irreparable DNA double-strand breaks was applied to the biological endpoint of chromosomal aberrations. The model explains irreparable DNA double-strand breaks in terms of break clustering in DNA subunits. The model predicts quite good chromosomal aberrations in gamma- and X-ray irradiated V79 cells and human lymphocytes. In the case of α-particle irradiation the presumption had to be made, that only the cells with indirect events in the nucleus (due to delta-electrons) reach the metaphase and are analysed. With the help of this model we are able to explain the peculiar effectiveness of ultrasoft C-X-rays in human lymphocytes. In addition, an interpretation of experiments with accelerated and spatially correlated particles is given. (author)

  8. 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

  9. An aromatic sensor with aversion to damaged strands confers versatility to DNA repair.

    Directory of Open Access Journals (Sweden)

    Olivier Maillard

    2007-04-01

    Full Text Available It was not known how xeroderma pigmentosum group C (XPC protein, the primary initiator of global nucleotide excision repair, achieves its outstanding substrate versatility. Here, we analyzed the molecular pathology of a unique Trp690Ser substitution, which is the only reported missense mutation in xeroderma patients mapping to the evolutionary conserved region of XPC protein. The function of this critical residue and neighboring conserved aromatics was tested by site-directed mutagenesis followed by screening for excision activity and DNA binding. This comparison demonstrated that Trp690 and Phe733 drive the preferential recruitment of XPC protein to repair substrates by mediating an exquisite affinity for single-stranded sites. Such a dual deployment of aromatic side chains is the distinctive feature of functional oligonucleotide/oligosaccharide-binding folds and, indeed, sequence homologies with replication protein A and breast cancer susceptibility 2 protein indicate that XPC displays a monomeric variant of this recurrent interaction motif. An aversion to associate with damaged oligonucleotides implies that XPC protein avoids direct contacts with base adducts. These results reveal for the first time, to our knowledge, an entirely inverted mechanism of substrate recognition that relies on the detection of single-stranded configurations in the undamaged complementary sequence of the double helix.

  10. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Junk, Michael; Salzwedel, Judy; Sindlinger, Thilo; Bürkle, Alexander; Moreno-Villanueva, Maria

    2014-01-01

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  11. Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

    Science.gov (United States)

    He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

    2013-11-01

    Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

  12. SIRT participates at DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi Yong; Joeng, Jae Min; Lee, Kee Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Park, Gil Hong [College of Medicine, Korea University, Seoul (Korea, Republic of)

    2009-05-15

    Sir2 maintains genomic stability in multiple ways in yeast. As a NAD{sup +}-dependent histone deacetylase, Sir2 has been reported to control chromatin silencing. In both budding yeast and Drosophila, overexpression of Sir2 extends life span. Previous reports have also demonstrated that Sir2 participate at DNA damage repair. A protein complex containing Sir2 has been reported to translocate to DNA double-strand breaks. Following DNA damage response, SIRT1 deacetylates p53 protein and attenuates its ability as a transcription factor. Consequently, SIRT1 over-expression increases cell survival under DNA damage inducing conditions. These previous observations mean a possibility that signals generated during the process of DNA repair are delivered through SIRT1 to acetylated p53. We present herein functional evidence for the involvement of SIRT1 in DNA repair response to radiation. In addition, this modulation of DNA repair activity may be connected to deacetylation of MRN proteins.

  13. Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2

    DEFF Research Database (Denmark)

    Leshets, Michael; Ramamurthy, Dharanidharan; Lisby, Michael

    2018-01-01

    One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the......One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ......) are the main DSB repair pathways. Fumarase is a mitochondrial enzyme which functions in the tricarboxylic acid cycle. Intriguingly, the enzyme can be readily detected in the cytosolic compartment of all organisms examined, and we have shown that cytosolic fumarase participates in the DNA damage response...

  14. 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.

  15. Primary DNA damage in chrome-plating workers.

    Science.gov (United States)

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

    2003-06-30

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

  16. Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

    Science.gov (United States)

    Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V

    2015-03-04

    Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

  17. Double strand DNA breaks response in Huntington´s disease

    Czech Academy of Sciences Publication Activity Database

    Šolc, Petr; Valášek, Jan; Rausová, Petra; Juhásová, Jana; Juhás, Štefan; Motlík, Jan

    2015-01-01

    Roč. 78, Suppl 2 (2015), s. 15-15 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA MŠk ED2.1.00/03.0124; GA MŠk(CZ) 7F14308 Institutional support: RVO:67985904 Keywords : Huntington ´s disease * DNA damage * double strand DNA breaks Subject RIV: FH - Neurology

  18. SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

    DEFF Research Database (Denmark)

    McCord, Ronald A; Michishita, Eriko; Hong, Tao

    2009-01-01

    -PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

  19. Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

    Science.gov (United States)

    Rezaee, Mohammad; Sanche, Léon; Hunting, Darel J

    2013-03-01

    The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing

  20. Enzymatic induction of DNA double-strand breaks in γ-irradiated Escherichia coli K-12

    International Nuclear Information System (INIS)

    Bonura, T.; Smith, K.C.; Kaplan, H.S.

    1975-01-01

    The polA1 mutation increases the sensitivity of E. coli K-12 to killing by γ-irradiation in air by a factor of 2.9 and increases the yield of DNA double-strand breaks by a factor of 2.5. These additional DNA double-strand breaks appear to be due to the action of nucleases in the polA1 strain rather than to the rejoining of radiation-induced double-strand breaks in the pol + strain. This conclusion is based upon the observation that γ-irradiation at 3 0 did not affect the yield of DNA double-strand breaks in the pol + strain, but decreased the yield in the polA1 strain by a factor of 2.2. Irradiation of the polA1 strain at 3 0 followed by incubation at 3 0 for 20 min before plating resulted in approximately a 1.5-fold increase in the D 0 . The yield of DNA double-strand breaks was reduced by a factor of 1.5. The pol + strain, however, did not show the protective effect of the low temperature incubation upon either survival or DNA double-strand breakage. We suggest that the increased yield of DNA double-strand breaks in the polA 1 strain may be the result of the unsuccessful excision repair of ionizing radiation-induced dna base damage

  1. Alkali-labile sites and post-irradiation effects in single-stranded DNA induced by H radicals

    International Nuclear Information System (INIS)

    Lafleur, M.V.M.; Heuvel, N. van; Woldhuis, J.; Loman, H.

    1978-01-01

    Single-stranded phiX174 DNA in aqueous solutions has been irradiated in the absence of oxygen, under conditions in which H radicals react with the DNA. It was shown that H radical reactions result in breaks, which contribute approximately 10 per cent inactivation. Further, two types of alkali-labile sites were formed. One was lethal and gave rise to single-strand breaks by alkali and was most probably identical with post-irradiation heat damage and contributed about 33 per cent to the inactivation mentioned above. The other consisted of non-lethal damage, partly dihydropyrimidine derivatives, and was converted to lethal damage by alkali. This followed from experiments in which the DNA was treated with osmium-tetroxide, which oxidized thymine to 5,6-dihydroxydihydrothymine. Treatment with alkali of this DNA gave the same temperature dependence as found for the non-lethal alkali-labile sites in irradiated DNA. A similar temperature dependence was found for dihydrothymine and irradiated pyrimidines with alkali. (author)

  2. Repair pathways for heavy ion-induced complex DNA double strand breaks

    International Nuclear Information System (INIS)

    Yajima, Hirohiko; Nakajima, Nakako; Hirakawa, Hirokazu; Murakami, Takeshi; Okayasu, Ryuichi; Fujimori, Akira

    2012-01-01

    DNA double strand break (DSB) induced by ionizing radiation (IR) is a deleterious damage leading to cell death and genome instability if not properly repaired. It is well known that DSB is repaired by two major pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). It is also known that NHEJ is dominant throughout the cell cycle after X- or gamma-ray irradiation in mammalian cells, Meanwhile, it is thought that heavy-ion radiation (e.g., carbon-ions, iron-ions) gives rise to clustered DNA damages consisting of not only strand breaks but also aberrant bases in the vicinity of DSBs (complex DSBs). Our previous work suggested that the efficiency of NHEJ is diminished for repair of complex DSBs induced by heavy-ion radiation. We thought that this difficulty in NHEJ process associated with heavy ion induced complex DNA damage might be extended to HR process in cells exposed to heavy ions. In order to find out if this notion is true or not, exposed human cells to X-rays and heavy-ions, and studied HR associated processes at the molecular level. Our result indicates that complex DSBs induced by heavy ions effectively evoke DNA end resection activity during the HR process. Together with our results, a relevant recent progress in the field of DNA DSB repair will be discussed. (author)

  3. 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.

  4. Genetic and biochemical identification of a novel single-stranded DNA binding complex in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Amy eStroud

    2012-06-01

    Full Text Available Single-stranded DNA binding proteins play an essential role in DNA replication and repair. They use oligosaccharide-binding folds, a five-stranded ß-sheet coiled into a closed barrel, to bind to single-stranded DNA thereby protecting and stabilizing the DNA. In eukaryotes the single-stranded DNA binding protein is known as replication protein A (RPA and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed single-stranded DNA-binding protein (SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3 exist in operons with a novel gene specific to Euryarchaeota, this gene encodes a protein that we have termed rpa-associated protein (RPAP. The rpap genes encode proteins belonging to COG3390 group and feature oligosaccharide-binding folds, suggesting that they might cooperate with RPA in binding to single-stranded DNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only ∆rpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins. We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA binding complex that is unique to Euryarchaeota.

  5. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

    International Nuclear Information System (INIS)

    Moreau, P.L.

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

  6. Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Kuo, W.L.; Meyn, R.E.; Haidle, C.W.

    1984-01-01

    The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity

  7. WE-E-BRE-11: New Method to Simulate DNA Damage Using Ionization Cross-Sections and a Geometrical Nucleosome Model

    International Nuclear Information System (INIS)

    Pater, P; Seuntjens, J; El Naqa, I

    2014-01-01

    Purpose: To obtain probability distributions of various DNA damage types as a function of the incident electron kinetic energy. Methods: Using Geant4-DNA electron ionization cross-sections, we calculated path length distributions for electrons of energies between 10 eV and 1 MeV, defined as the length between two subsequent interactions. These path lengths were then convolved with probability distributions for the creation of same-strand damage, opposite-strand damage, clustered damage, isolated damage, and same DNA strand target damage. These probability distributions of DNA damage were obtained by a Monte Carlo routine calculating probabilities of interaction in DNA targets inside a nucleosome geometrical model. Results represent the probability of a secondary electron, initially created inside a DNA strand target, of undergoing its next interaction: (1) in the opposite strand (DSB), (2) in the same strand (SSB+), (3) in either the opposite or same-strand (clustered), (4) in the same DNA target (multiple-hit) or (5) more than 10 base pairs away (isolated). Results: Electrons with kinetic energy between 50 and 250 eV have a maximal probability of creating DSB, SSB+, clustered damage and multiple-hits in the same target The probabilities for these damage patterns have values of 2.5%, 4.3%, 6.7% and 5.4%, respectively. Isolated damage is most probable between 700 eV to 900 eV with a probability of 0.2%. Conclusion: We obtained DNA damage probability distributions as a function of electron incident energy. We showed that electrons with kinetic energies between 50 and 250 eV have the highest probability of producing complex forms of DNA damage (DSB, SSB+). We also showed that a double ionization within the same DNA target is the most frequent outcome occurring 5% of the time. It is expected that electron slowing down spectra can be convolved with our formalism to calculate source specific DNA damage patterns. Research grants from governments of Canada and Quebec. PP

  8. Radiolysis of chromatin extracted from cultured mammalian cells: production of alkali-labile strand damage in DNA

    International Nuclear Information System (INIS)

    Mee, L.K.; Adelstein, S.J.; Stein, G.

    1978-01-01

    Chromatin has been isolated from cultured Chinese-hamster lung fibroblasts as an expanded aqueous gel. The DNA in isolated chromatin has been examined by sedimentation on alkaline sucrose gradients. The average molecular weight of the DNA has been determined to be 50 million. γ -irradiation of isolated chromatin degraded the DNA to lower molecular weight. The yield of single-strand breaks in the DNA was 0.02 single-strand breaks per krad-10 6 dalton, calculated from a dose-range of 1 to 400 krad and covering a DNA molecular weight range of 2 x 10 7 to 1.4 x 10 5 . There was a considerable difference in the efficiency of the formation of single-strand breaks in DNA irradiated as isolated chromatin compared with chromatin irradiated in whole cells before isolation. For isolated chromatin, values of 6 eV per break have been calculated compared with about 80 eV per break for chromatin irradiated in whole cells, which suggest a large contribution from indirect action by aqueous radicals in isolated chromatin. (author)

  9. Assessment of DNA damage induced by terrestrial UV irradiation of dried bloodstains: forensic implications.

    Science.gov (United States)

    Hall, Ashley; Sims, Lynn M; Ballantyne, Jack

    2014-01-01

    Few publications have detailed the nature of DNA damage in contemporary (i.e. non-ancient) dried biological stains. The chief concern, from a forensic standpoint, is that the damage can inhibit polymerase-mediated primer extension, ultimately resulting in DNA typing failure. In the work described here, we analyzed the effects of UVA and UVB irradiation on cell-free solubilized DNA, cell-free dehydrated DNA and dehydrated cellular DNA (from bloodstains). After UV exposure ranging from 25 J cm(-2) to 1236 J cm(-2), we assayed for the presence of bipyrimidine photoproducts (BPPPs), oxidative lesions and strand breaks, correlating the damage with the inhibition of STR profiling. Subsequent to irradiation with either UVA and UVB, the incidence of BPPPs, oxidative products and strand breaks were observed in decreasing quantities as follows: cell-free solubilized DNA>cell-free dehydrated DNA>bloodstain DNA. UVA irradiation did not result in even the partial loss of a STR profile in any sample tested. Somewhat different results were observed after genetic analysis of UVB exposed samples, in that the ability to produce a complete STR profile was affected earliest in bloodstain DNA, next in cell-free solubilized DNA and not at all in cell-free dehydrated DNA. Therefore, it is likely that other types of damage contributed to allele-drop-out in these samples but remained undetected by our assays, whereby the endonucleases did not react with the lesions or the presence of the lesions was masked by strand breaks. Under the conditions of the study, strand breaks appeared to be the predominant types of damage that ultimately resulted in DNA typing failure from physiological stains, although some evidence suggested oxidative damage may have played a role as well. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. 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

  11. 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.

  12. Assembly and function of DNA double-strand break repair foci in mammalian cells

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2010-01-01

    DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks...

  13. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

  14. Modulation of X ray DNA damage by SR-2508 +/- buthionine sulfoximine

    International Nuclear Information System (INIS)

    Kinsella, T.J.; Dobson, P.P.; Russo, A.; Mitchell, J.B.; Fornace, A.J. Jr.

    1986-01-01

    It has been demonstrated that glutathione (GSH) depletion with buthionine sulfoximine (BSO) potentiates SR-2508 radiosensitization in hypoxic cells. We have measured the effect of SR-2508 alone, BSO alone, and combined treatment on radiation-induced DNA strand breaks in hypoxic V79 cells using alkaline and neutral elution. DNA base damage recognized by a damage specific endonuclease from M. luteus was also studied. Hypoxic irradiation markedly reduces the efficiency of single-strand (SSB) and double-strand breaks (DSB) to 10-20% compared to oxic irradiation. Hypoxia had less effect on the efficiency of base damage (ESS). BSO treatment alone, resulting in GSH depletion to less than 5% of controls, had little effect of hypoxic-SSB, DSB, and ESS. SR-2508 (5 mM) treatment alone in hypoxic cells increased the number of SSB, DSB and ESS to approximately half of that resulting from oxic irradiation. However, the combination of BSO and SR-2508 in hypoxic cells resulted in SSB and DSB comparable to oxic irradiation. This combined treatment resulted in less effect on ESS. We conclude that the observed hypoxic radiosensitization, using clonogenic survival assays with combined BSO-SR-2508, correlates with our results assessing DNA strand breaks and base damage

  15. Molecular characterization of a complex site-specific radiation-induced DNA double-strand break

    International Nuclear Information System (INIS)

    Datta, K.; Dizdaroglu, M.; Jaruga, P.; Neumann, R.D.; Winters, T.A.

    2003-01-01

    Radiation lethality is a function of radiation-induced DNA double-strand breaks (DSB). Current models propose the lethality of a DSB to be a function of its structural complexity. We present here for the first time a map of damage associated with a site-specific double-strand break produced by decay of 125 I in a plasmid bound by a 125 I-labeled triplex forming oligonucleotide ( 125 I-TFO). The E. coli DNA repair enzymes, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-DNA glycosylase (Fpg), which recognize AP sites, and pyrimidine and purine base damage respectively, were used as probes in this study. 125 I-TFO bound plasmid was incubated with and without DMSO at -80 deg C for 1 month. No significant difference in DSB yield was observed under these conditions. A 32 base pair fragment from the upstream side of the decay site was isolated by restriction digestion and enzymatically probed to identify damage sites. Endo IV treatment of the 5'-end labeled upper strand indicated clustering of AP sites within 3 bases downstream and 7 bases upstream of the targeted base. Also, repeated experiments consistently detected an AP site 4 bases upstream of the 125 Itarget base. This was further supported by complementary results with the 3'-end labeled upper strand. Endo IV analysis of the lower strand also shows clustering of AP sites near the DSB end. Endo III and Fpg probing demonstrated that base damage is also clustered near the targeted break site. DSBs produced in the absence of DMSO displayed a different pattern of enzyme sensitive damage than those produced in the presence of DMSO. Identification of specific base damage types within the restriction fragment containing the DSB end was achieved with GC/MS. Base damage consisted of 8-hydroguanine, 8-hydroxyadenine, and 5-hydroxycytosine. These lesions were observed at relative yields of 8-hydroguanine and 5-hydroxycytosine to 8-hydroxyadenine of 7.4:1 and 4.7:1, respectively, in the absence

  16. Measurement of intracellular DNA double-strand break induction and rejoining along the track of carbon and neon particle beams in water

    International Nuclear Information System (INIS)

    Heilmann, Johannes; Taucher-Scholz, Gisela; Haberer, Thomas; Scholz, Michael; Kraft, Gerhard

    1996-01-01

    Purpose: The study was aimed at the measurement of effect-depth distributions of intracellularly induced DNA damage in water as tissue equivalent after heavy ion irradiation with therapy particle beams. Methods and Materials: An assay involving embedding of Chinese hamster ovary (CHO-K1) cells in large agarose plugs and electrophoretic elution of radiation induced DNA fragments by constant field gel electrophoresis was developed. Double-strand break production was quantified by densitometric analysis of DNA-fluorescence after staining with ethidium-bromide and determination of the fraction of DNA eluted out of the agarose plugs. Intracellular double-strand break induction and the effect of a 3 h rejoining incubation were investigated following irradiation with 250 kV x-rays and 190 MeV/u carbon- and 295 MeV/u neon-ions. Results and Conclusion: While the DNA damage induced by x-irradiation decreased continuously with penetration depth, a steady increase in the yield of double-strand breaks was observed for particle radiation, reaching distinct maxima at the position of the physical Bragg peaks. Beyond this, the extent of radiation damage dropped drastically. From comparison of DNA damage and calculated dose profiles, relative biological efficiencies (RBEs) for both double-strand break induction and unrejoined strand breaks after 3 h were determined. While RBE for the induction of DNA double-strand breaks decreased continuously with penetration depth, RBE maxima greater than unity were found with carbon- and neon-ions for double-strand break rejoining near the maximum range of the particles. The method presented here allows for a fast and accurate determination of depth profiles of relevant radiobiological effects for mixed particle fields in tissue equivalent

  17. Double strand breaks in DNA in vivo and in vitro after 60Co-γ-irradiation

    International Nuclear Information System (INIS)

    Huelsewede, J.W.

    1985-01-01

    The questions of what the correlation is between double strand breaks in DNA in the cell and lethal radiation damage and by means of which possible mechanisms DNA double strand breaks could occur were studied. E. coli served as test system. In addition to this the molecular weight of the DNA from irradiated E. coli as a function of the radiation dose under various conditions was measured. This data was compared on the one hand to the survival of the cell and on the other hand to the formation of DNA double strand breaks in an aqueous buffer system, which in its ionic characteristics was similar to cell fluids. (orig./MG) [de

  18. Symposium cellular response to DNA damage the role of poly(ADP-ribose) poly(ADP-ribose) in the cellular response to DNA damage

    International Nuclear Information System (INIS)

    Berger, N.A.

    1985-01-01

    Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Inhibitors of Poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of Poly(ADP-ribose) persists and the activated enzyme is capable of totaly consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of Poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest

  19. Mechanisms of DNA damage by the tumor promoter and progressor benzoyl peroxide

    International Nuclear Information System (INIS)

    Swauger, J.E.; Dolan, P.M.; Zweier, J.L.; Kensler, T.W.

    1990-01-01

    Benzoyl peroxide (BzPO), a tumor promoter and progressor in mouse skin, produces strand breaks in DNA of exposed cells. Previously we have reported that the metabolism of BzPO in keratinocytes proceeds via the initial cleavage of the peroxide bond, yielding benzoyloxyl radicals which, in turn, can fragment to form phenyl radicals and carbon dioxide. Benzoic acid, the product of hydrogen abstraction by the benzoyloxyl radical, is the major stable metabolite of BzPO produced by keratinocytes. In the present study we have examined the capacity of BzPO to generate strand scissions in φX-174 plasmid DNA. DNA damage was dose-dependent over a concentration range of 10-1000 μM BzPO and was dependent on the presence of copper but not other transition state metals. By contrast, benzoic acid did not produce DNA damage in this system. The inclusion of spin trapping agents (PBN, DBNBS), radical scavenging agents (Nal, GSH), or the copper chelator o-phenanthroline in incubations was found to significantly reduce the extent of DNA damage. Electron paramagnetic resonance spectroscopy studies suggested that the primary radical trapped was the benzoyloxyl radical, implying a role for this radical in the generation of the observed DNA damage. Collectively these observations suggest BzPO may be activated to DNA damaging intermediates in keratinocytes via metal-catalyzed cleavage of the peroxide bond resulting in the formation of the benzoyloxyl radical. Covalent modification of DNA was not observed when [ 14 C]BzPO was incubated with calf thymus DNA in the presence of copper. Overall, these results suggest that BzPO induces DNA damage via benzoyloxyl radical mediated proton abstraction from the DNA strand and the adduct formation with DNA is unlikely to occur

  20. hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

    International Nuclear Information System (INIS)

    Berglund, Fredrik M.; Clarke, Paul R.

    2009-01-01

    Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

  1. 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.

  2. 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

  3. 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

  4. Quantitation of the repair of gamma-radiation-induced double-strand DNA breaks in human fibroblasts

    International Nuclear Information System (INIS)

    Woods, W.G.

    1981-01-01

    The quantitation and repair of double-strand DNA breaks in human fibroblasts has been determined using a method involving the nondenaturing elution of DNA from a filter. DNA from cells from two human fibroblast lines exposed to γ-radiation from 0 to 10000 rad showed increasing retention on a filter with decreasing radiation dose, and the data suggest a linear relationship between double-strand breaks induced and radiation dose. The ability of normal human fibroblasts to repair double-strand breaks with various doses of radiation was demonstrated, with a tsub(1/2) of 10 min for repair of 5000 rad exposure and 39 min for repair of 10000 rad damage. The kinetics of the DNA rejoining were not linear and suggest that, as in the repair of single-strand breaks, both an initial fast and a later slow mechanism may be involved. (Auth.)

  5. Clustered DNA damage induced by proton and heavy ion irradiation

    International Nuclear Information System (INIS)

    Davidkova, M.; Pachnerova Brabcova, K; Stepan, V.; Vysin, L.; Sihver, L.; Incerti, S.

    2014-01-01

    Ionizing radiation induces in DNA strand breaks, damaged bases and modified sugars, which accumulate with increasing density of ionizations in charged particle tracks. Compared to isolated DNA damage sites, the biological toxicity of damage clusters can be for living cells more severe. We investigated the clustered DNA damage induced by protons (30 MeV) and high LET radiation (C 290 MeV/u and Fe 500 MeV/u) in pBR322 plasmid DNA. To distinguish between direct and indirect pathways of radiation damage, the plasmid was irradiated in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The goal of the contribution is the analysis of determined types of DNA damage in dependence on radiation quality and related contribution of direct and indirect radiation effects. The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers. (authors)

  6. Evaluation of the neutral comet assay for detection of alpha-particle induced DNA-double-strand-breaks

    International Nuclear Information System (INIS)

    Hofbauer, Daniela

    2010-01-01

    Aim of this study was to differentiate DNA-double-strand-breaks from DNA-single-strand-breaks on a single cell level, using the comet assay after α- and γ-irradiation. Americium-241 was used as a alpha-irradiation-source, Caesium-137 was used for γ-irradiation. Because of technical problems with both the neutral and alkaline comet assay after irradiation of gastric cancer cells and human lymphocytes, no definite differentiation of DNA-damage was possible.

  7. Plasma induced DNA damage: Comparison with the effects of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Lazović, S.; Maletić, D.; Puač, N.; Malović, G.; Petrović, Z. Lj. [Institute of Physics, University of Belgrade, Pregrevica 118, 11080 Belgrade (Serbia); Leskovac, A.; Filipović, J.; Joksić, G. [Department of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, 11001 Belgrade (Serbia)

    2014-09-22

    We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of the treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.

  8. SAMHD1 Sheds Moonlight on DNA Double-Strand Break Repair.

    Science.gov (United States)

    Cabello-Lobato, Maria Jose; Wang, Siyue; Schmidt, Christine Katrin

    2017-12-01

    SAMHD1 (sterile α motif and histidine (H) aspartate (D) domain-containing protein 1) is known for its antiviral activity of hydrolysing deoxynucleotides required for virus replication. Daddacha et al. identify a hydrolase-independent, moonlighting function of SAMHD1 that facilitates homologous recombination of DNA double-strand breaks (DSBs) by promoting recruitment of C-terminal binding protein interacting protein (CTIP), a DNA-end resection factor, to damaged DNA. These findings could benefit anticancer treatment. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Method for detecting DNA strand breaks in mammalian cells using the Deinococcus radiodurans PprA protein

    International Nuclear Information System (INIS)

    Satoh, Katsuya; Wada, Seiichi; Kikuchi, Masahiro; Funayama, Tomoo; Narumi, Issay; Kobayashi, Yasuhiko

    2006-01-01

    In a previous study, we identified the novel protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans. In this study, we focussed on the ability of PprA protein to recognize and bind to double-stranded DNA carrying strand breaks, and attempted to visualize radiation-induced DNA strand breaks in mammalian cultured cells by employing PprA protein using an immunofluorescence technique. Increased PprA protein binding to CHO-K1 nuclei immediately following irradiation suggests the protein is binding to DNA strand breaks. By altering the cell permeabilization conditions, PprA protein binding to CHO-K1 mitochondria, which is probably resulted from DNA strand break immediately following irradiation, was also detected. The method developed and detailed in this study will be useful in evaluating DNA damage responses in cultured cells, and could also be applicable to genotoxic tests in the environmental and pharmaceutical fields

  10. Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB).

    Science.gov (United States)

    van Loon, Barbara; Samson, Leona D

    2013-03-01

    Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair. Copyright © 2012. Published by Elsevier B.V.

  11. 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.

  12. 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.

  13. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

    International Nuclear Information System (INIS)

    Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

    2004-01-01

    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

  14. Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line

    International Nuclear Information System (INIS)

    Bouffler, S.D.; Jha, B.; Johnson, R.T.

    1990-01-01

    DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate

  15. 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)

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

    Science.gov (United States)

    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 bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo. PMID:22941641

  17. Mechanisms of free radical-induced damage to DNA.

    Science.gov (United States)

    Dizdaroglu, Miral; Jaruga, Pawel

    2012-04-01

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

  18. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

    Science.gov (United States)

    Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

    2016-01-01

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

  19. 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.

  20. 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.

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

    Science.gov (United States)

    Panier, Stephanie; Durocher, Daniel

    2013-10-01

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

  2. Excess single-stranded DNA inhibits meiotic double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Rebecca Johnson

    2007-11-01

    Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

  3. Evaluating In Vitro DNA Damage Using Comet Assay.

    Science.gov (United States)

    Lu, Yanxin; Liu, Yang; Yang, Chunzhang

    2017-10-11

    DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

  4. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

    Science.gov (United States)

    Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

    2016-03-04

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Connecting localized DNA strand displacement reactions

    Science.gov (United States)

    Mullor Ruiz, Ismael; Arbona, Jean-Michel; Lad, Amitkumar; Mendoza, Oscar; Aimé, Jean-Pierre; Elezgaray, Juan

    2015-07-01

    Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions.Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/C5NR02434J

  6. 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.)

  7. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways.

    Science.gov (United States)

    Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil; Shuman, Stewart; Glickman, Michael S

    2011-01-01

    Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways. © 2010 Blackwell Publishing Ltd.

  8. Correlations of DNA strand breaks and their repair with cell survival following acute exposure to mercury(II) and X-rays

    International Nuclear Information System (INIS)

    Cantoni, O.; Costa, M.

    1983-01-01

    Alkaline elution analysis demonstrates that both HgCl 2 and X-rays result in a rapid induction of DNA single-strand breaks at acutely cytotoxic doses (HgCl 2 , 25-100 microM for 60 min; X-rays, 150-600 rads) in cultured Chinese hamster ovary cells. Cytotoxicity, as measured by cell-plating efficiency, correlates linearly with the level of DNA breakage induced by both agents (HgCl 2 , r . 0.97; X-rays, r . 0.99), although a substantial difference in axis intercepts of the two linear regression lines indicates that a higher level of DNA damage was required by X-rays as compared with HgCl 2 to produce an equivalent level of cell killing. DNA damage induced by X-rays was rapidly repaired such that within 1 hr following treatment the elution rate of DNA from treated cells resembled that obtained in untreated cultures. In contrast, DNA damage after Hg 2+ insult was not repaired, and further damage was evident following a similar 1-hr recovery period. Addition of noncytotoxic, non-DNA-damaging concentrations of HgCl 2 (10 microM) to cells 15-45 min following treatment with X-rays greatly inhibited the repair of the DNA strand breaks. Thus, although both HgCl 2 and X-rays induce rapid and striking single-strand breaks in the DNA, persistence of Hg 2+ in the cell can inhibit the repair of these breaks. The inhibition of DNA repair by HgCl 2 may explain why this agent is not severely mutagenic or carcinogenic despite its ability to induce an X-ray-like DNA damage and why a lower level of mercury-induced DNA damage, compared with that induced by X-rays, was required to produce an equivalent level of cell death

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

    NARCIS (Netherlands)

    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

  10. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    Science.gov (United States)

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  11. Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites

    Science.gov (United States)

    Mortusewicz, Oliver; Roth, Wera; Li, Na; Cardoso, M. Cristina; Meisterernst, Michael; Leonhardt, Heinrich

    2008-01-01

    The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identified as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae. To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fluorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fluorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is independent of poly(ADP-ribosyl)ation and γH2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or facilitate the subsequent steps of DNA repair. PMID:19047459

  12. Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

    International Nuclear Information System (INIS)

    Lopez-Larraza, Daniel M.; Padron, Juan; Ronci, Natalia E.; Vidal Rioja, Lidia A.

    2006-01-01

    Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 o C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells

  13. Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

    Energy Technology Data Exchange (ETDEWEB)

    Lopez-Larraza, Daniel M. [IMBICE, C.C. 403, 1900 La Plata (Argentina)]. E-mail: danielop@imbice.org.ar; Padron, Juan [IMBICE, C.C. 403, 1900 La Plata (Argentina); Ronci, Natalia E. [IMBICE, C.C. 403, 1900 La Plata (Argentina); Vidal Rioja, Lidia A. [IMBICE, C.C. 403, 1900 La Plata (Argentina)

    2006-08-30

    Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 {sup o}C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.

  14. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  15. 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

  16. Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

    Science.gov (United States)

    Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

    2009-05-01

    Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

  17. Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

    Science.gov (United States)

    Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

    2018-01-01

    DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

  18. Molecular dynamics simulation of a DNA containing a single strand break

    Energy Technology Data Exchange (ETDEWEB)

    Yamaguchi, H.; Siebers, G.; Furukawa, A.; Otagiri, N.; Osman, R

    2002-07-01

    Molecular dynamics simulations were performed for a dodecamer DNA containing a single strand break (SSB), which has been represented by a 3'-OH deoxyribose and 5'-OH phosphate in the middle of the strand. Molecular force field parameters of the 5'-OH phosphate region were determined from an ab initio calculation at the HF/6-31G level using the program package GAMESS. The DNA was placed in a periodic boundary box with water molecules and Na+ counter-ions to produce a neutralised system. After minimisation, the system was heated to 300 K, equilibrated and a production run at constant NTP was executed for 1 ns using AMBER 4.1. Snapshots of the SSB-containing DNA and a detailed analysis of the equilibriated average structure revealed surprisingly small conformational changes compared to normal DNA. However, dynamic properties calculated using the essential dynamics method showed some features that may be important for the recognition of this damage by repair enzymes. (author)

  19. Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Ifigeneia V. Mavragani

    2017-07-01

    Full Text Available Cellular effects of ionizing radiation (IR are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs, single strand breaks (SSBs and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1 repair resistant, increasing genomic instability (GI and malignant transformation and (2 can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity. Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.

  20. 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)

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

    Science.gov (United States)

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

    1994-03-01

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

  2. Carcinogen-induced damage to DNA

    International Nuclear Information System (INIS)

    Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

    1979-01-01

    Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

  3. DNA damage by carbonyl stress in human skin cells

    International Nuclear Information System (INIS)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L.

    2003-01-01

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N ε -(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress

  4. DNA damage by carbonyl stress in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L

    2003-01-28

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the {alpha}-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N{sup {epsilon}}-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

  5. Formation of double-strand breaks in DNA of γ-irradiated bacteria depending on the function of fast repair processes of DNA single-strand breaks

    International Nuclear Information System (INIS)

    Petrov, S.I.; Gaziev, A.I.

    1980-01-01

    The formation of double-strand breaks in DNA of γ-irradiated ( 60 Co)Ex coli bacteria depending on the function of fast repair processes of DNA single-strand breaks, is investigated. The profiles of sedimentation of DNA Ex coli cells, irradiated at 0-2 deg C in the salt medium and in EDTA-borate buffer, are presented. It is shown that when irradiating cells in EDTA-borate buffer, the output of single- and double strand breaks in DNA is much higher than in the case of their irradiation in the minimum salt medium. The dependence of output of single-strand and double-strand breaks depending on the radiatier doze of E coli cells in the salt medium and EDTA-borate buffer, is studied. The supposition is made on the presence of a regulative interaction between the accumulation of DNA single-breaks and their repair with the formation of double-strand breaks. The functionating of fast and superfast repair processes considerably affects the formation of double-strand breaks in DNA of a bacterium cell. A considerable amount of double-breaks registered immediately after irradiation forms due to a close position of single-strand breaks on the opposite DNA strands

  6. Nick translation detection in situ of cellular DNA strand break induced by radiation

    International Nuclear Information System (INIS)

    Maehara, Y.; Anai, H.; Kusumoto, T.; Sakaguchi, Y.; Sugimachi, K.

    1989-01-01

    DNA strand break in HeLa cells induced by radiation was detected using the in situ nick translation method. The cells were exposed to radiation of 3, 6, 12, 18, and 24 Gy in Lab-Tek tissue culture chamber/slides and were fixed with ethanol/acetic acid on the slide glass. The break sites in DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [ 3 H]-labeled dTTP. Autoradiographic observation was made of the level of break sites in the DNA. The DNA strand break appeared even with a 3 Gy exposure, increased 8.6 times at 24 Gy compared with the control cells, and this level correlated reciprocally to change in cell viability. This nick translation method provides a rapid in situ assay for determining radiation-induced DNA damage of cultured cells, in a semi-quantitative manner

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

    Science.gov (United States)

    Mullenders, Leon

    2015-12-01

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

  8. Role of Rad54, Rad54b and Snm1 in DNA damage repair

    NARCIS (Netherlands)

    J. Wesoly (Joanna)

    2003-01-01

    textabstractThe aim of this thesis is to investigate the function of a number of genes involved in mammalian DNA damage repair, in particular in repair of DNA double-strand breaks (DSBs). Among a large number of different damages that can be introduced to DNA, DSBs are especially toxic. If

  9. 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.

  10. Radiation-induced DNA damage in tumors and normal tissues. II. Influence of dose, residual DNA damage and physiological factors in oxygenated cells

    International Nuclear Information System (INIS)

    Zhang, H.; Wheeler, K.T.

    1994-01-01

    Detection and quantification of hypoxic cells in solid tumors is important for many experimental and clinical situations. Several laboratories, including ours, have suggested that assays which measure radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elution assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must not be formed to any great extent in irradiated oxygenated cells, and the formation and repair of strand breaks and DPCs in oxygenated cells must not be modified appreciably by physiological factors (e.g., temperature, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In one set of experiments, oxygenated 9L cells in tissue culture, subcutaneous 9L tumors and rat cerebella were irradiated with doses of 15 or 50 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated exponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose of 15 Gy at 37 or 20 degrees C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of strand breaks or the formation of DPCs in oxygenated 9L cells. These results support the use of this DNA damage assay for the detection and quantification of hypoxic cells in solid tumors. 27 refs., 5 tabs

  11. Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

    International Nuclear Information System (INIS)

    Galli, A.; Schiestl, R.H.

    1998-01-01

    Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both γ-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas γ-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBsbut not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. (author)

  12. Damage of DNA by radiation and it's recovery, 3

    International Nuclear Information System (INIS)

    Narita, Noboru; Matsuura, Tomio; Sato, Hiroyuki.

    1974-01-01

    The damage and recovery of DNA was investigated by the incorporation of thymine derivatives (DHT, I trans, II trans, cis and glycol) into exponentially growing Tetrahymena cells. The strain employed was Tetrahymena pyriformis, Variety I, mating type IV. It is well known that these thymine derivatives are induced in vivo by radiation. The in vivo damage of DNA induced by radiation, and its recovery, were confirmed experimentally by means of gradient separation of sucrose density and by analytical ultra centrifugation (UVC). The recovery of DNA, its excision repair and its recombinational repair were compared with the recovery of Bacillus subtilis whose recovery kinetics were already known. 1) The damage of DNA was more sensitive to glycol than to II trans and cis. On the other hand, DHT is not sensitive for breaking DNA strand. 2) In its recovery damaged DNA was no more sensitive to glycol than to hhp as was true for Bacillus subtilis. (author)

  13. Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation

    Directory of Open Access Journals (Sweden)

    Britta Muster

    2017-02-01

    Full Text Available Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in vivo in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.

  14. Control of DNA strand displacement kinetics using toehold exchange.

    Science.gov (United States)

    Zhang, David Yu; Winfree, Erik

    2009-12-02

    DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

  15. Equilibrious Strand Exchange Promoted by DNA Conformational Switching

    Science.gov (United States)

    Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

    2013-01-01

    Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

  16. Variation in normal and tumor tissue sensitivity of mice to ionizing radiation-induced DNA strand breaks in vivo

    International Nuclear Information System (INIS)

    Meyn, R.E.; Jenkins, W.T.

    1983-01-01

    The efficiency of DNA strand break formation in normal and tumor tissues of mice was measured using the technique of alkaline elution coupled with a microfluorometric determination of DNA. This methodology allowed measurement of the DNA strand breaks produced in tissues irradiated in vivo with doses of radiation comparable to those used in radiotherapy (i.e., 1.0 gray) without the necessity for the cells to be dividing and incorporating radioactive precursors to label the DNA. The results showed that substantial differences existed among various tissues in terms of the amount of DNA strand break damage produced for a given dose of radiation. Of the normal tissues, the most breaks were produced in bone marrow and the least were produced in gut. Furthermore, strand break production was relatively inefficient in the tumor compared to the normal tissues. The efficiency of DNA strand break formation measured in the cells from the tissues irradiated in vitro was much more uniform and considerably greater than that measured in vivo, suggesting that the normal tissues in the animal may be radiobiologically hypoxic

  17. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

    Science.gov (United States)

    Kanno, Shin-ichiro; Kuzuoka, Hiroyuki; Sasao, Shigeru; Hong, Zehui; Lan, Li; Nakajima, Satoshi; Yasui, Akira

    2007-01-01

    DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine–tyrosine–arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with γH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes. PMID:17396150

  18. Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks

    Science.gov (United States)

    Stante, Maria; Minopoli, Giuseppina; Passaro, Fabiana; Raia, Maddalena; Vecchio, Luigi Del; Russo, Tommaso

    2009-01-01

    Fe65 is a binding partner of the Alzheimer's β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites. PMID:19282473

  19. On the consistency of Monte Carlo track structure DNA damage simulations

    Energy Technology Data Exchange (ETDEWEB)

    Pater, Piotr, E-mail: piotr.pater@mail.mcgill.ca; Seuntjens, Jan; El Naqa, Issam [McGill University, Montreal, Quebec H3G 1A4 (Canada); Bernal, Mario A. [Instituto de Fisica Gleb Wataghin, Universidade Estadual de Campinas, Campinas 13083-859 (Brazil)

    2014-12-15

    Purpose: Monte Carlo track structures (MCTS) simulations have been recognized as useful tools for radiobiological modeling. However, the authors noticed several issues regarding the consistency of reported data. Therefore, in this work, they analyze the impact of various user defined parameters on simulated direct DNA damage yields. In addition, they draw attention to discrepancies in published literature in DNA strand break (SB) yields and selected methodologies. Methods: The MCTS code Geant4-DNA was used to compare radial dose profiles in a nanometer-scale region of interest (ROI) for photon sources of varying sizes and energies. Then, electron tracks of 0.28 keV–220 keV were superimposed on a geometric DNA model composed of 2.7 × 10{sup 6} nucleosomes, and SBs were simulated according to four definitions based on energy deposits or energy transfers in DNA strand targets compared to a threshold energy E{sub TH}. The SB frequencies and complexities in nucleosomes as a function of incident electron energies were obtained. SBs were classified into higher order clusters such as single and double strand breaks (SSBs and DSBs) based on inter-SB distances and on the number of affected strands. Results: Comparisons of different nonuniform dose distributions lacking charged particle equilibrium may lead to erroneous conclusions regarding the effect of energy on relative biological effectiveness. The energy transfer-based SB definitions give similar SB yields as the one based on energy deposit when E{sub TH} ≈ 10.79 eV, but deviate significantly for higher E{sub TH} values. Between 30 and 40 nucleosomes/Gy show at least one SB in the ROI. The number of nucleosomes that present a complex damage pattern of more than 2 SBs and the degree of complexity of the damage in these nucleosomes diminish as the incident electron energy increases. DNA damage classification into SSB and DSB is highly dependent on the definitions of these higher order structures and their

  20. On the consistency of Monte Carlo track structure DNA damage simulations

    International Nuclear Information System (INIS)

    Pater, Piotr; Seuntjens, Jan; El Naqa, Issam; Bernal, Mario A.

    2014-01-01

    Purpose: Monte Carlo track structures (MCTS) simulations have been recognized as useful tools for radiobiological modeling. However, the authors noticed several issues regarding the consistency of reported data. Therefore, in this work, they analyze the impact of various user defined parameters on simulated direct DNA damage yields. In addition, they draw attention to discrepancies in published literature in DNA strand break (SB) yields and selected methodologies. Methods: The MCTS code Geant4-DNA was used to compare radial dose profiles in a nanometer-scale region of interest (ROI) for photon sources of varying sizes and energies. Then, electron tracks of 0.28 keV–220 keV were superimposed on a geometric DNA model composed of 2.7 × 10 6 nucleosomes, and SBs were simulated according to four definitions based on energy deposits or energy transfers in DNA strand targets compared to a threshold energy E TH . The SB frequencies and complexities in nucleosomes as a function of incident electron energies were obtained. SBs were classified into higher order clusters such as single and double strand breaks (SSBs and DSBs) based on inter-SB distances and on the number of affected strands. Results: Comparisons of different nonuniform dose distributions lacking charged particle equilibrium may lead to erroneous conclusions regarding the effect of energy on relative biological effectiveness. The energy transfer-based SB definitions give similar SB yields as the one based on energy deposit when E TH ≈ 10.79 eV, but deviate significantly for higher E TH values. Between 30 and 40 nucleosomes/Gy show at least one SB in the ROI. The number of nucleosomes that present a complex damage pattern of more than 2 SBs and the degree of complexity of the damage in these nucleosomes diminish as the incident electron energy increases. DNA damage classification into SSB and DSB is highly dependent on the definitions of these higher order structures and their implementations. The authors

  1. 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

  2. DNA turnover and strand breaks in Escherichia coli

    International Nuclear Information System (INIS)

    Hanawalt, P.; Grivell, A.; Nakayama, H.

    1975-01-01

    The extent of DNA turnover has been measured in a dnaB mutant of Escherichia coli, temperature sensitive for semiconservative DNA replication. At the nonpermissive temperature about 0.02 percent of the deoxynucleotides in DNA are exchanged per generation period. This turnover rate is markedly depressed in the presence of rifampicin. During thymine starvation strand breaks accumulate in the DNA of E. coli strains that are susceptible to thymineless death. Rifampicin suppresses the appearance of these breaks, consistent with our hypothesis that transcription may be accompanied by repairable single-strand breaks in DNA. DNA turnover is enhanced severalfold in strands containing 5-bromodeoxyuridine in place of thymidine, possibly because the analog (or the deoxyuridine, following debromination) is sometimes recognized and excised

  3. DNA damage-inducible transcripts in mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

    1988-01-01

    Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

  4. A model capturing novel strand symmetries in bacterial DNA

    International Nuclear Information System (INIS)

    Sobottka, Marcelo; Hart, Andrew G.

    2011-01-01

    Highlights: → We propose a simple stochastic model to construct primitive DNA sequences. → The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. → The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. → We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. → We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

  5. 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)

  6. ATM signaling and genomic stability in response to DNA damage

    International Nuclear Information System (INIS)

    Lavin, Martin F.; Birrell, Geoff; Chen, Philip; Kozlov, Sergei; Scott, Shaun; Gueven, Nuri

    2005-01-01

    DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed

  7. Possible role(s) of nuclear matrix and DNA loop organization in fixation or repair of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Malyapa, R.S.; Wright, W.D.; Roti Roti, J.L.

    1995-01-01

    DNA double-strand breaks produced by ionizing radiation are considered to be a critical radiation-induced lesion responsible, in part, for cell killing. However, the manner in which structures within the nucleus involving DNA organization contribute to the balance between fixation or repair of these critical lesions remains largely obscure. The repair process requires both functional enzymes and substrate availability, i.e., access to and orientation of damage sites. Therefore, the ability to repair damaged DNA could be influenced not only by DNA integrity but also by the spatial organization of DNA. Therefore, the authors investigated the possibility that radiation-induced DNA damage differentially affects DNA supercoiling ability in cells of differing radiosensitivities using radioresistant and radiosensitive mutants of different origins. This study was also designed to determine if differences in the composition of the nuclear matrix exist between cell lines of each origin. Results from these studies indicate that differences in the composition of the nuclear matrix proteins and DNA stability might be related to intrinsic radiation resistance

  8. Fragmentation in DNA double-strand breaks

    International Nuclear Information System (INIS)

    Wei Zhiyong; Suzhou Univ., Suzhou; Zhang Lihui; Li Ming; Fan Wo; Xu Yujie

    2005-01-01

    DNA double strand breaks are important lesions induced by irradiations. Random breakage model or quantification supported by this concept is suitable to analyze DNA double strand break data induced by low LET radiation, but deviation from random breakage model is more evident in high LET radiation data analysis. In this work we develop a new method, statistical fragmentation model, to analyze the fragmentation process of DNA double strand breaks. After charged particles enter the biological cell, they produce ionizations along their tracks, and transfer their energies to the cells and break the cellular DNA strands into fragments. The probable distribution of the fragments is obtained under the condition in which the entropy is maximum. Under the approximation E≅E 0 + E 1 l + E 2 l 2 , the distribution functions are obtained as exp(αl + βl 2 ). There are two components, the one proportional to exp(βl 2 ), mainly contributes to the low mass fragment yields, the other component, proportional to exp(αl), decreases slowly as the mass of the fragments increases. Numerical solution of the constraint equations provides parameters α and β. Experimental data, especially when the energy deposition is higher, support the statistical fragmentation model. (authors)

  9. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    Science.gov (United States)

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

    2015-09-01

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

  11. Electrophoresis examination of strand breaks in plasmid DNA induced by low-energy nitrogen ion irradiation

    International Nuclear Information System (INIS)

    Zhao Yong; Tan Zheng; Du Yanhua; Qiu Guanying

    2003-01-01

    To study the effect on plasmid DNA of heavy ion in the energy range of keV where nuclear stopping interaction becomes more important or even predominant, thin film of plasmid pGEM-3Zf(-) DNA was prepared on aluminum surface and irradiated in vacuum ( -3 Pa) by low-energy nitrogen ions with energy of 30 keV (LET=285 keV/μm) at various fluence ranging from 2 x 10 10 to 8.2 x 10 13 ions/cm 2 . DNA strand breaks were analyzed by neutral electrophoresis followed by quantification with image analysis software. Low-energy nitrogen ion irradiation induced single-, double- and multiple double-strand breaks (DSB) and multiple DSB as the dominating form of DNA damages. Moreover, the linear fluence-response relationship at a low fluence range suggests that DSBs are induced predominantly by single ion track. However, strand break production is limited to a short range in the irradiated samples

  12. 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

  13. Delayed repair of radiation induced clustered DNA damage: Friend or foe?

    International Nuclear Information System (INIS)

    Eccles, Laura J.; O'Neill, Peter; Lomax, Martine E.

    2011-01-01

    A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a 'friend', leading to cell killing in tumour cells or as a 'foe', resulting in the formation of mutations and genetic instability in normal tissue.

  14. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Ian Hare

    2016-01-01

    Full Text Available Mesenchymal stem cells (MSCs are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs, the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16 at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

  15. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

    Science.gov (United States)

    Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A; Howell, David; Gibson, Laura F

    2016-01-01

    Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

  16. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...... and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...

  17. DNA-imprinted polymer nanoparticles with monodispersity and prescribed DNA-strand patterns

    Science.gov (United States)

    Trinh, Tuan; Liao, Chenyi; Toader, Violeta; Barłóg, Maciej; Bazzi, Hassan S.; Li, Jianing; Sleiman, Hanadi F.

    2018-02-01

    As colloidal self-assembly increasingly approaches the complexity of natural systems, an ongoing challenge is to generate non-centrosymmetric structures. For example, patchy, Janus or living crystallization particles have significantly advanced the area of polymer assembly. It has remained difficult, however, to devise polymer particles that associate in a directional manner, with controlled valency and recognition motifs. Here, we present a method to transfer DNA patterns from a DNA cage to a polymeric nanoparticle encapsulated inside the cage in three dimensions. The resulting DNA-imprinted particles (DIPs), which are 'moulded' on the inside of the DNA cage, consist of a monodisperse crosslinked polymer core with a predetermined pattern of different DNA strands covalently 'printed' on their exterior, and further assemble with programmability and directionality. The number, orientation and sequence of DNA strands grafted onto the polymeric core can be controlled during the process, and the strands are addressable independently of each other.

  18. Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline

    Energy Technology Data Exchange (ETDEWEB)

    Petry, T.W.; Bowden, G.T.; Huxtable, R.J.; Sipes, I.G.

    1984-04-01

    Hepatic DNA damage induced by the pyrrolizidine alkaloid monocrotaline was evaluated following i.p. administration to adult male Sprague-Dawley rats. Animals were treated with various doses ranging upward from 5 mg/kg, and hepatic nuclei were isolated 4 hr later. Hepatic nuclei were used as the DNA source in all experiments. DNA damage was characterized by the alkaline elution technique. A mixture of DNA-DNA interstrand cross-links and DNA-protein cross-links was induced. Following an injection of monocrotaline, 30 mg/kg i.p., DNA-DNA interstrand cross-linking reached a maximum within 12 hr or less and thereafter decreased over a protracted period of time. By 96 hr postadministration, the calculated cross-linking factor was no longer statistically different from zero. No evidence for the induction of DNA single-strand breaks was observed, although the presence of small numbers of DNA single-strand breaks could have been masked by the overwhelming predominance of DNA cross-links. These DNA cross-links may be related to the hepatocarcinogenic, hepatotoxic, and/or antimitotic effects of monocrotaline.

  19. 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.

  20. Induction of DNA strand breaks in 14C-labelled cells

    International Nuclear Information System (INIS)

    Sundell-Bergman, S.; Johanson, K.J.

    1979-01-01

    Chinese hamster cells grown in vitro were labelled with 14 C-thymidine for 18 hours and after 3 hours in non-radioactive medium they were stored at 0 0 C for various periods ( 1 to 12 hours). During this treatment a number of DNA strand breaks were induced by 14 C decay which were not repaired at 0 0 C. The number of DNA strand breaks was determined using the DNA unwinding technique. At 0.5-1 dpm per cell a detectable number of DNA strand breaks were found. Treatment for six hours (1 dpm per cell) reduced the percentage of double-stranded DNA from 80 to 70%, corresponding to about 750 DNA strand breaks per cell. The rejoining of DNA strand breaks was studied after treatment for 12 hours at 0 0 C followed by incubation of the cells for various periods at 37 0 C. Most of the DNA strand breaks induced by 14 C decay at 0 0 C were repaired after incubation at 37 0 C for 15 minutes. Assuming an absorbed dose of 1.8 mGy per 14 C decay to the cell nucleus an RBE value close to 1 was found for internal irradiation from 14 C decay as compared with 60 Co-gamma irradiation. (author)

  1. A Novel Computational Method to Reduce Leaky Reaction in DNA Strand Displacement

    Directory of Open Access Journals (Sweden)

    Xin Li

    2015-01-01

    Full Text Available DNA strand displacement technique is widely used in DNA programming, DNA biosensors, and gene analysis. In DNA strand displacement, leaky reactions can cause DNA signals decay and detecting DNA signals fails. The mostly used method to avoid leakage is cleaning up after upstream leaky reactions, and it remains a challenge to develop reliable DNA strand displacement technique with low leakage. In this work, we address the challenge by experimentally evaluating the basic factors, including reaction time, ratio of reactants, and ion concentration to the leakage in DNA strand displacement. Specifically, fluorescent probes and a hairpin structure reporting DNA strand are designed to detect the output of DNA strand displacement, and thus can evaluate the leakage of DNA strand displacement reactions with different reaction time, ratios of reactants, and ion concentrations. From the obtained data, mathematical models for evaluating leakage are achieved by curve derivation. As a result, it is obtained that long time incubation, high concentration of fuel strand, and inappropriate amount of ion concentration can weaken leaky reactions. This contributes to a method to set proper reaction conditions to reduce leakage in DNA strand displacement.

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

    Directory of Open Access Journals (Sweden)

    Concetta Cuozzo

    2007-07-01

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

  3. Seasonal and PAH impact on DNA strand-break levels in gills of transplanted zebra mussels.

    Science.gov (United States)

    Michel, Cécile; Bourgeault, Adeline; Gourlay-Francé, Catherine; Palais, Frédéric; Geffard, Alain; Vincent-Hubert, Françoise

    2013-06-01

    Genotoxicity endpoints are useful tools to biomonitor the physicochemical and biological quality of aquatic ecosystems. A caging study on the freshwater bivalve Dreissena polymorpha was planned to run over four seasons in the Seine River basin in order to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. Three sites were chosen along the Seine River, one upstream from Paris and two downstream, corresponding to a chemical gradient of water contamination. The DNA strand break (comet assay) and chromosomal damage (micronucleus test) were measured in caged mussels at each site and in winter, spring and summer, along with PAH water contamination, PAH bioaccumulation, the mussel condition index (CI), the gonado-somatic index (GSI) and the filtration rate (FR). The level of DNA strand break measured in winter was low and increased in spring, concomitantly with FR and GSI. Over the same period, micronucleus (MN) frequency and PAH bioaccumulation decreased significantly in caged mussels, with both parameters positively correlated to each other. DNA strand-break levels and MN frequencies showed inter-site variations corresponding to the chemical contamination gradient. These two genotoxicity endpoints usefully complement each other in field studies. These results show that the MN test and comet assay, when applied to gill cells of caged zebra mussels, are sensitive tools for freshwater genotoxicity monitoring. Copyright © 2013. Published by Elsevier Inc.

  4. Sub-ensemble monitoring of DNA strand displacement using multiparameter single-molecule FRET

    OpenAIRE

    Baltierra Jasso, Laura; Morten, Michael; Magennis, Steven William

    2018-01-01

    Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constan...

  5. 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)

  6. Ionizing-radiation induced DNA double-strand breaks: A direct and indirect lighting up

    International Nuclear Information System (INIS)

    Vignard, Julien; Mirey, Gladys; Salles, Bernard

    2013-01-01

    The occurrence of DNA double-strand breaks (DSBs) induced by ionizing radiation has been extensively studied by biochemical or cell imaging techniques. Cell imaging development relies on technical advances as well as our knowledge of the cell DNA damage response (DDR) process. The DDR involves a complex network of proteins that initiate and coordinate DNA damage signaling and repair activities. As some DDR proteins assemble at DSBs in an established spatio-temporal pattern, visible nuclear foci are produced. In addition, post-translational modifications are important for the signaling and the recruitment of specific partners at damaged chromatin foci. We briefly review here the most widely used methods to study DSBs. We also discuss the development of indirect methods, using reporter expression or intra-nuclear antibodies, to follow the production of DSBs in real time and in living cells

  7. Epidermal growth factor stimulating reparation of γ-ray-induced single-strand breaks predominantly in untranscribed DNA of HeLa cells

    International Nuclear Information System (INIS)

    Igusheva, O.A.; Bil'din, V.N.; Zhestyanikov, V.D.

    1994-01-01

    Considerable evidence suggest that genomic DNA undergoes reparation unevenly because of different transcription activities of its particular sequence. It is highly probably that transcriptional factors are necessary for postion stages of excision reparation and for reparation of single-strand DNA breaks caused by ionizing radiation. There is evidence suggesting that DNA lesions inflicted by γ-radiation is preferentially initiated in transcribed rather than in untranscribed DNA species. This paper looks at the relationship between stimulatory effect of epidermal growth factor (EGF) on reparation of single-strand DNA breaks and reparation of the damage done to active and inert fragments of chromatin. The results show that EGF stimulates reparation of single-strand DNA breaks induced by γ-radiation more effectively in untranscribed than in transcribed DNA. 13 refs., 1 fig., 1 tab

  8. DNA Damage Following Pulmonary Exposure by Instillation to Low Doses of Carbon Black (Printex 90) Nanoparticles in Mice

    DEFF Research Database (Denmark)

    Kyjovska, Zdenka O.; Jacobsen, Nicklas R.; Saber, Anne T.

    2015-01-01

    of 0.67, 2, 6, and 162 mu g Printex 90 NPCB and vehicle. Cellular composition and protein concentration was evaluated in BAL fluid as markers of inflammatory response and cell damage. DNA strand breaks in BAL cells, lung, and liver tissue were assessed using the alkaline comet assay. The pulmonary...... the comet assay. We interpret the increased DNA strand breaks occurring following these low exposure doses of NPCB as DNA damage caused by primary genotoxicity in the absence of substantial inflammation, cell damage, and acute phase response. Environ. Mol. Mutagen. 56:41-49, 2015. (c) 2014 The Authors...

  9. Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

    Directory of Open Access Journals (Sweden)

    Simon Roux

    2016-12-01

    Full Text Available Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA viral genomes captured in quantitative viral metagenomes (viromes. This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation. Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5% of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

  10. Spectroscopic approaches to study DNA damage induced in genome exposed to ionizing radiation and its enzymatic repair

    International Nuclear Information System (INIS)

    Yokoya, Akinari; Fujii, Kentaro; Oka, Toshitaka; Watanabe, Ritsuko

    2012-01-01

    Recent progress on spectroscopic study on physicochemical process of DNA damage induction will be reported. It has been predicted by computer track simulation studies that complex DNA damage, so called clustered DNA damage sites, is produced along the tack particularly of high Linear Energy Transfer (LET) ions. The clustered DNA damage, consisting of two or more isolated lesions such as single strand breaks or nucleobase lesions, is thought to compromise DNA repair enzymes. We have revealed that the nucleobase lesions produced by He 2+ ion impact to simple model DNA (plasmid) are hardly processed by base excision repair enzymes (E. coli DNA glycosylases). Using the third generation synchrotron radiation facility (SPring-8), we have studied unpaired electron species or desorbed ions as intermediates of DNA damage using an EPR apparatus or mass spectrometer installed in the soft X-ray beamline in SPring-8. These aspects are compared with the yields of final products of single- and double-strand breaks and base lesions revealed biochemical techniques. Models of complex DNA damage induction will be proposed considering various modification factors of the damage induction, ionization of valence and inner-shell electrons, OH radicals, hydration layer and the impact of secondary electrons. (author)

  11. DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawłowska

    2017-07-01

    Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

  12. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    Science.gov (United States)

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  13. Stem cell identity and template DNA strand segregation.

    Science.gov (United States)

    Tajbakhsh, Shahragim

    2008-12-01

    The quest for stem cell properties to distinguish their identity from that of committed daughters has led to a re-investigation of the notion that DNA strands are not equivalent, and 'immortal' DNA strands are retained in stem cells whereas newly replicated DNA strands segregate to the differentiating daughter cell during mitosis. Whether this process occurs only in stem cells, and also in all tissues, remains unclear. That individual chromosomes can be also partitioned non-randomly raises the question if this phenomenon is related to the immortal DNA hypothesis, and it underscores the need for high-resolution techniques to observe these events empirically. Although initially postulated as a mechanism to avoid DNA replication errors, alternative views including epigenetic regulation and sister chromatid silencing may provide insights into this process.

  14. Torsional regulation of hRPA-induced unwinding of double-stranded DNA

    NARCIS (Netherlands)

    De Vlaminck, I.; Vidic, I.; Van Loenhout, M.T.J.; Kanaar, R.; Lebbink, J.H.G.; Dekker, C.

    2010-01-01

    All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the

  15. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    International Nuclear Information System (INIS)

    Arimoto-Kobayashi, Sakae; Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko

    2010-01-01

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in φX174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  16. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    Energy Technology Data Exchange (ETDEWEB)

    Arimoto-Kobayashi, Sakae [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan); Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan)

    2010-09-10

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in {phi}X174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  17. Strand displacement activated peroxidase activity of hemin for fluorescent DNA sensing.

    Science.gov (United States)

    Wang, Quanbo; Xu, Nan; Gui, Zhen; Lei, Jianping; Ju, Huangxian; Yan, Feng

    2015-10-07

    To efficiently regulate the catalytic activity of the peroxidase mimic hemin, this work designs a double-stranded DNA probe containing an intermolecular dimer of hemin, whose peroxidase activity can be activated by a DNA strand displacement reaction. The double-stranded probe is prepared by annealing two strands of hemin labelled DNA oligonucleotides. Using the fluorescent oxidation product of tyramine by H2O2 as a tracing molecule, the low peroxidase activity of the hemin dimer ensures a low fluorescence background. The strand displacement reaction of the target DNA dissociates the hemin dimer and thus significantly increases the catalytic activity of hemin to produce a large amount of dityramine for fluorescence signal readout. Based on the strand displacement regulated peroxidase activity, a simple and sensitive homogeneous fluorescent DNA sensing method is proposed. The detection can conveniently be carried out in a 96-well plate within 20 min with a detection limit of 0.18 nM. This method shows high specificity, which can effectively distinguish single-base mismatched DNA from perfectly matched target DNA. The DNA strand displacement regulated catalytic activity of hemin has promising application in the determination of various DNA analytes.

  18. 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

  19. ZTF-8 interacts with the 9-1-1 complex and is required for DNA damage response and double-strand break repair in the C. elegans germline.

    Directory of Open Access Journals (Sweden)

    Hyun-Min Kim

    2014-10-01

    Full Text Available Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR and DSB repair (DSBR within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.

  20. DNA damage and radical reactions: Mechanistic aspects, formation in cells and repair studies

    International Nuclear Information System (INIS)

    Cadet, J.; Ravanat, J.L.; Carell, T.; Cellai, L.; Chatgilialoglu, Ch.; Gimisis, Th.; Miranda, M.; O'Neill, P.; Robert, M.

    2008-01-01

    Several examples of oxidative and reductive reactions of DNA components that lead to single and tandem modifications are discussed in this review. These include nucleophilic addition reactions of the one-electron oxidation-mediated guanine radical cation and the one-electron reduced intermediate of 8-bromo-purine 2'-de-oxy-ribo-nucleosides that give rise to either an oxidizing guanine radical or related 5',8-cyclo-purine nucleosides. In addition, mechanistic insights into the reductive pathways involved in the photolyase induced reversal of cyclo-buta-cli-pyrimidine and pyrimidine (6-4) pyrimidone photoproducts are provided. Evidence for the occurrence and validation in cellular DNA of (OH) · radical degradation pathways of guanine that have been established in model systems has been gained from the accurate measurement of degradation products. Relevant information on biochemical aspects of the repair of single and clustered oxidatively generated damage to DNA has been gained from detailed investigations that rely on the synthesis of suitable modified probes. Thus the preparation of stable carbocyclic derivatives of purine nucleoside containing defined sequence oligonucleotides has allowed detailed crystallographic studies of the recognition step of the base damage by enzymes implicated in the base excision repair (BER) pathway. Detailed insights are provided on the BER processing of non-double strand break bi-stranded clustered damage that may consist of base lesions, a single strand break or abasic sites and represent one of the main deleterious classes of radiation-induced DNA damage. (authors)

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

    Science.gov (United States)

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

    1989-01-01

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

  2. Delayed repair of radiation induced clustered DNA damage: Friend or foe?

    Science.gov (United States)

    Eccles, Laura J.; O’Neill, Peter; Lomax, Martine E.

    2011-01-01

    A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue. PMID:21130102

  3. Human Xip1 (C2orf13) is a novel regulator of cellular responses to DNA strand breaks

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Fugger, Kasper; Danielsen, Jannie Rendtlew

    2007-01-01

    DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we...

  4. Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

    2016-01-01

    Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA......-binding proteins have previously been found to be phosphorylated on tyrosine and arginine residues. While tyrosine phosphorylation was shown to enhance the DNA-binding properties of SsbA, arginine phosphorylation was not functionally characterized.Materials and methods: We used mass spectrometry analysis to detect...... phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation...

  5. Investigation of DNA strand breaks induced by 7Li and 12C ions

    International Nuclear Information System (INIS)

    Sui Li; Zhao Kui; Ni Meinan; Guo Jiyu; Luo Hongbing; Mei Junping; Lu Xiuqin; Zhou Ping

    2004-01-01

    Deoxyribonucleic acid (DNA) is an important biomacromolecule. It is a carrier of genetic information and a critical target for radiobiological effects. Numerous lesions have been identified in irradiated DNA. DNA double strand breaks (DSBs) are considered as the most important initial damage of all biological effects induced by ionizing radiation. In this experiment, DNA DSBs induced by heavy ions in the early period were investigated with atomic force microscopy (AFM). Choosing 7 Li and 12 C heavy ions with different LET values delivered by HI-13 tandem accelerator respectively, purified plasmid DNA samples in aqueous solution were irradiated at different doses. AFM was used for nanometer-level-structure analysis of DNA damage induced by these two kinds of heavy ions. Measurement of the DNA fragment lengths was accomplished with the Scion Image analyzed soft-ware. Change laws of three forms of DNA, supercoils, open circular and linear form as dose increased were obtained. Distributed function of DNA fragment length was also obtained, and fitted with Tsallis entropy statistical theory. (author)

  6. Detection and Repair of Ionizing Radiation-Induced DNA Double Strand Breaks: New Developments in Nonhomologous End Joining

    International Nuclear Information System (INIS)

    Wang, Chen; Lees-Miller, Susan P.

    2013-01-01

    DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation

  7. Detection and Repair of Ionizing Radiation-Induced DNA Double Strand Breaks: New Developments in Nonhomologous End Joining

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chen [Departments of Biochemistry and Molecular Biology and Oncology, and Southern Alberta Cancer Research Institute, University of Calgary, Calgary (Canada); Lees-Miller, Susan P., E-mail: leesmill@ucalgary.ca [Departments of Biochemistry and Molecular Biology and Oncology, and Southern Alberta Cancer Research Institute, University of Calgary, Calgary (Canada)

    2013-07-01

    DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation.

  8. Double-Strand DNA Break Repair in Mycobacteria.

    Science.gov (United States)

    Glickman, Michael S

    2014-10-01

    Discontinuity of both strands of the chromosome is a lethal event in all living organisms because it compromises chromosome replication. As such, a diversity of DNA repair systems has evolved to repair double-strand DNA breaks (DSBs). In part, this diversity of DSB repair systems has evolved to repair breaks that arise in diverse physiologic circumstances or sequence contexts, including cellular states of nonreplication or breaks that arise between repeats. Mycobacteria elaborate a set of three genetically distinct DNA repair pathways: homologous recombination, nonhomologous end joining, and single-strand annealing. As such, mycobacterial DSB repair diverges substantially from the standard model of prokaryotic DSB repair and represents an attractive new model system. In addition, the presence in mycobacteria of a DSB repair system that can repair DSBs in nonreplicating cells (nonhomologous end joining) or when DSBs arise between repeats (single-strand annealing) has clear potential relevance to Mycobacterium tuberculosis pathogenesis, although the exact role of these systems in M. tuberculosis pathogenesis is still being elucidated. In this article we will review the genetics of mycobacterial DSB repair systems, focusing on recent insights.

  9. Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

    Science.gov (United States)

    Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

    2015-03-31

    Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

  10. 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.

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

    Science.gov (United States)

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

    2016-07-26

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

  12. In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication.

    Science.gov (United States)

    Schauer, Grant; Finkelstein, Jeff; O'Donnell, Mike

    2017-09-20

    The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their 'correct' strands, as well as quality control mechanisms that evict polymerases when they associate with an 'incorrect' strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.

  13. Detection of UVR-induced DNA damage in mouse epidermis in vivo using alkaline elution

    International Nuclear Information System (INIS)

    Kinley, J.S.; Moan, J.; Brunborg, G.

    1995-01-01

    Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320-400 nm) or indirectly by UVB (280-320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). (Author)

  14. Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

    Science.gov (United States)

    Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

    2018-03-05

    Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

    Science.gov (United States)

    Choi, Jun-Hyuk; Lindsey-Boltz, Laura A; Kemp, Michael; Mason, Aaron C; Wold, Marc S; Sancar, Aziz

    2010-08-03

    ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

  16. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

    2014-01-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

  17. S1 nuclease from Aspergillus oryzae for the detection of DNA damage and repair in the gamma-irradiated intracerebral rat gliosarcoma 9L

    International Nuclear Information System (INIS)

    Gutin, P.H.; Hilton, J.; Fein, V.J.; Allen, A.E.; Walker, M.D.

    1977-01-01

    DNA damage and repair in a rat brain tumor following irradiation in vivo were measured by analysis of the rate of strand separation of the tumor DNA in alkali. Tumors were removed after irradiation and mechanically dissociated to a cellular suspension. Tumor cells were injected into alkali (pH 12) for 20 min at 22 0 C. The fraction of tumor DNA remaining double-stranded after this exposure to alkali was determined by its resistance to S 1 nuclease from Aspergillus oryzae. Double-stranded DNA remains (after enzyme exposure) acid-precipitable for fluorescent assay. The double-stranded fraction after exposure to alkali decreases with increasing radiation dose following first-order kinetics. DNA from tumors excised at intervals after irradiation showed a greater double-stranded fraction in alkali than that from tumors excised immediately, indicating repair of single-strand breaks. Repair of damage produced by 600 rad proceeded with a half-time of approximately 15 min

  18. Human telomeres are hypersensitive to UV-induced DNA Damage and refractory to repair.

    Directory of Open Access Journals (Sweden)

    Patrick J Rochette

    2010-04-01

    Full Text Available Telomeric repeats preserve genome integrity by stabilizing chromosomes, a function that appears to be important for both cancer and aging. In view of this critical role in genomic integrity, the telomere's own integrity should be of paramount importance to the cell. Ultraviolet light (UV, the preeminent risk factor in skin cancer development, induces mainly cyclobutane pyrimidine dimers (CPD which are both mutagenic and lethal. The human telomeric repeat unit (5'TTAGGG/CCCTAA3' is nearly optimal for acquiring UV-induced CPD, which form at dipyrimidine sites. We developed a ChIP-based technique, immunoprecipitation of DNA damage (IPoD, to simultaneously study DNA damage and repair in the telomere and in the coding regions of p53, 28S rDNA, and mitochondrial DNA. We find that human telomeres in vivo are 7-fold hypersensitive to UV-induced DNA damage. In double-stranded oligonucleotides, this hypersensitivity is a property of both telomeric and non-telomeric repeats; in a series of telomeric repeat oligonucleotides, a phase change conferring UV-sensitivity occurs above 4 repeats. Furthermore, CPD removal in the telomere is almost absent, matching the rate in mitochondria known to lack nucleotide excision repair. Cells containing persistent high levels of telomeric CPDs nevertheless proliferate, and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions. Utilizing a lesion-tolerance strategy rather than repair would prevent double-strand breaks at closely-opposed excision repair sites on opposite strands of a damage-hypersensitive repeat.

  19. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    International Nuclear Information System (INIS)

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-01-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA III ); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA III [URO-MSC(+)] and after subsequent removal of MMA III [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA III , URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA III . URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA III exposure, suggesting the presence of MMA III is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA III results in: the induction of DNA damage that remains elevated upon removal of MMA III ; increased levels of ROS that play a role in MMA III induced-DNA damage; and decreased PARP activity in the presence of MMA III .

  20. DNA double strand breaks in the acute phase after synchrotron pencilbeam irradiation

    International Nuclear Information System (INIS)

    Fernandez-Palomo, C; Trippel, M; Schroll, C; Nikkhah, G; Schültke, E; Bräuer-Krisch, E; Requardt, H; Bartzsch, S

    2013-01-01

    Introduction. At the biomedical beamline of the European Synchrotron Radiation Facility (ESRF), we have established a method to study pencilbeam irradiation in-vivoin small animal models. The pencilbeam irradiation technique is based on the principle of microbeam irradiation, a concept of spatially fractionated high-dose irradiation. Using γH2AX as marker, we followed the development of DNA double strand breaks over 48 hrs after whole brain irradiation with the pencilbeam technique. Method. Almost square pencilbeams with an individual size of 51 × 50 μm were produced with an MSC collimator using a step and shoot approach, while the animals were moved vertically through the beam. The center-to-center distance (ctc) was 400 μm, with a peak-to-valley dose ratio (PVDR) of about 400. Five groups of healthy adult mice received peak irradiation doses of either 330 Gy or 2,460 Gy and valley doses of 0.82 Gy and 6.15 Gy, respectively. Animals were sacrificed at 2, 12 and 48 hrs after irradiation. Results. DNA double strand breaks are observed in the path of the pencilbeam. The size of the damaged volume undergoes changes within the first 48 hours after irradiation. Conclusions. The extent of DNA damage caused by pencilbeam irradiation, as assessed by H2AX antibody staining, is dose- dependent

  1. The Effect of Basepair Mismatch on DNA Strand Displacement

    OpenAIRE

    Broadwater, D.?W.?Bo; Kim, Harold?D.

    2016-01-01

    DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single base pair mismatch. The apparent displacement rate varied si...

  2. Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakajima

    Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

  3. Cadmium inhibits repair of UV-, methyl methanesulfonate- and N-methyl-N-nitrosourea-induced DNA damage in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Fatur, Tanja; Lah, Tamara T.; Filipic, Metka

    2003-01-01

    The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl 2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl 2 , the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl 2 the MMS-induced DNA strand breaks accumulated during the first 2 h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl 2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair

  4. A single-strand specific lesion drives MMS-induced hyper-mutability at a double-strand break in yeast.

    Science.gov (United States)

    Yang, Yong; Gordenin, Dmitry A; Resnick, Michael A

    2010-08-05

    Localized hyper-mutability (LHM) can be important in evolution, immunity, and genetic diseases. We previously reported that single-strand DNA (ssDNA) can be an important source of damage-induced LHM in yeast. Here, we establish that the generation of LHM by methyl methanesulfonate (MMS) during repair of a chromosomal double-strand break (DSB) can result in over 0.2 mutations/kb, which is approximately 20,000-fold higher than the MMS-induced mutation density without a DSB. The MMS-induced mutations associated with DSB repair were primarily due to substitutions via translesion DNA synthesis at damaged cytosines, even though there are nearly 10 times more MMS-induced lesions at other bases. Based on this mutation bias, the promutagenic lesion dominating LHM is likely 3-methylcytosine, which is single-strand specific. Thus, the dramatic increase in mutagenesis at a DSB is concluded to result primarily from the generation of non-repairable lesions in ssDNA associated with DSB repair along with efficient induction of highly mutagenic ssDNA-specific lesions. These findings with MMS-induced LHM have broad biological implications for unrepaired damage generated in ssDNA and possibly ssRNA. Published by Elsevier B.V.

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

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xu; Ptasinska, Sylwia [Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Department of Physics, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Klas, Matej [Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Liu, Yueying [Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Sharon Stack, M. [Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 (United States)

    2013-06-10

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  7. Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

    International Nuclear Information System (INIS)

    Sprung, Carl N.; Vasireddy, Raja S.; Karagiannis, Tom C.; Loveridge, Shanon J.; Martin, Roger F.; McKay, Michael J.

    2010-01-01

    Purpose: The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods: We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results: We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions: These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

  8. Characterization of non-dimer DNA lesions and cellular damages caused by ultraviolet light

    International Nuclear Information System (INIS)

    Nakao, Kumi

    1989-01-01

    To understand the mechanisms of carcinogenicity and cytotoxicity induced by ultraviolet (UV) light, non-dimer DNA damages produced by near UV light (wave-length: 290∼320 nm) were examined by alkaline elution using Chinese hamster V-79 cells. UV exposure produced a dose-dependent induction of DNA single strand breaks and DNA-protein crosslinks. However, neither of these DNA lesions were repaired within a 24 hr incubation of the cells following UV exposure. Rather the number of these lesions increased. Also, UV exposure inhibited DNA and RNA synthesis. In addition, UV induced both cytotoxicity and chromosomal aberration. Electron spin resornance (ESR) studies showed that the exposure of cells to UV light resulted in the appearance of an ESR signal at -120degC. The roles of glutathione, vitamin E and vitamin B 2 , which were celluar antioxidant, on the induction of cytotoxicity by UV exposure were also examined. Pretreatment with vitamin E reduced the cytotoxicty caused by UV, whereas neither preteatment with vitamin B 2 nor the alteration of cellular gluthaione content affected the cytotoxicity. These results suggest that non-dimer DNA damages, such as DNA single strand breaks and DNA-protein crosslinks play an important role in inducing UV-carcinogenicity and UV-cytotoxicity, and that the mechanisms of these damages may be associated with the generation of free radicals. (author)

  9. Genetic and Biochemical Identification of a Novel Single-Stranded DNA-Binding Complex in Haloferax volcanii.

    Science.gov (United States)

    Stroud, Amy; Liddell, Susan; Allers, Thorsten

    2012-01-01

    Single-stranded DNA (ssDNA)-binding proteins play an essential role in DNA replication and repair. They use oligonucleotide/oligosaccharide-binding (OB)-folds, a five-stranded β-sheet coiled into a closed barrel, to bind to ssDNA thereby protecting and stabilizing the DNA. In eukaryotes the ssDNA-binding protein (SSB) is known as replication protein A (RPA) and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3) exist in operons with a novel gene specific to Euryarchaeota; this gene encodes a protein that we have termed RPA-associated protein (rpap). The rpap genes encode proteins belonging to COG3390 group and feature OB-folds, suggesting that they might cooperate with RPA in binding to ssDNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only Δrpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs). We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

  10. The Effect of Basepair Mismatch on DNA Strand Displacement.

    Science.gov (United States)

    Broadwater, D W Bo; Kim, Harold D

    2016-04-12

    DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single basepair mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data indicate that a single base pair mismatch in the invader stalls branch migration and displacement occurs via direct dissociation of the destabilized incumbent strand from the substrate strand. We combined both branch migration and direct dissociation into a model, which we term the concurrent displacement model, and used the first passage time approach to quantitatively explain the salient features of the observed relationship. We also introduce the concept of splitting probabilities to justify that the concurrent model can be simplified into a three-step sequential model in the presence of an invader mismatch. We expect our model to become a powerful tool to design DNA-based reaction schemes with broad functionality. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Current topics in DNA double-strand break repair

    International Nuclear Information System (INIS)

    Kobayashi, Junya; Takata, Minoru; Iwabuchi, Kuniyoshi; Miyagawa, Kiyoshi; Sonoda, Eiichiro; Suzuki, Keiji; Tauchi, Hiroshi

    2008-01-01

    DNA double strand break (DSB) is one of the most critical types of damage which is induced by ionizing radiation. In this review, we summarize current progress in investigations on the function of DSB repair-related proteins. We focused on recent findings in the analysis of the function of proteins such as 53BP1, histone H2AX, Mus81-Eme1, Fanc complex, and UBC13, which are found to be related to homologous recombination repair or to non-homologous end joining. In addition to the function of these proteins in DSB repair, the biological function of nuclear foci formation following DSB induction is discussed. (author)

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

    Science.gov (United States)

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

    2018-05-01

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

  13. Vegetables and PUFA-rich plant oil reduce DNA strand breaks in individuals with type 2 diabetes

    DEFF Research Database (Denmark)

    Müllner, Elisabeth; Brath, Helmut; Pleifer, Simone

    2013-01-01

    SCOPE: Type 2 diabetes is a multifactorial disease associated with increased oxidative stress, which may lead to increased DNA damage. The aim of this study was to investigate the effect of a healthy diet on DNA oxidation in diabetics and nondiabetics. METHODS AND RESULTS: Seventy-six diabetic...... and 21 nondiabetic individuals participated in this study. All subjects received information about the benefits of a healthy diet, while subjects randomly assigned to the intervention group received additionally 300 g of vegetables and 25 mL PUFA-rich plant oil per day. DNA damage in mononuclear cells...... increase in plasma antioxidant concentrations. Diabetic individuals of the intervention group showed a significant reduction in HbA1c and DNA strand breaks. Levels of HbA1c were also improved in diabetics of the information group, but oxidative damage to DNA was not altered. Urinary 8-oxodG and 8-oxo...

  14. DNA damage in lens epithelium of cataract patients in vivo and ex vivo.

    Science.gov (United States)

    Øsnes-Ringen, Oyvind; Azqueta, Amaia O; Moe, Morten C; Zetterström, Charlotta; Røger, Magnus; Nicolaissen, Bjørn; Collins, Andrew R

    2013-11-01

    DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

  15. 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

  16. 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.

  17. Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

    International Nuclear Information System (INIS)

    Okui, T.; Endoh, D.; Arai, S.; Isogai, E.; Hayashi, M.

    1996-01-01

    The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells

  18. Damages induced in lambda phage DNA by enzyme-generated triplet acetone

    International Nuclear Information System (INIS)

    Menck, C.F.; Cabral Neto, J.B.; Gomes, R.A.; Faljoni-Alario, A.

    1985-01-01

    Exposure of lambda phage to triplet acetone, generated during the aerobic oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only in alkaline conditions, so true breaks were not observed. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed. (Author) [pt

  19. Study on DNA Damage Induced by Neon Beam Irradiation in Saccharomyces Cerevisiae

    International Nuclear Information System (INIS)

    Lu Dong; Li Wenjian; Wu Xin; Wang Jufang; Ma Shuang; Liu Qingfang; He Jinyu; Jing Xigang; Ding Nan; Dai Zhongying; Zhou Jianping

    2010-01-01

    Yeast strain Saccharomyces cerevisiae was irradiated with different doses of 85 MeV/u 20 Ne 10+ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks (DSBs) and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T→G and T→C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.

  20. Study on DNA Damage Induced by Neon Beam Irradiation in Saccharomyces Cerevisiae

    Science.gov (United States)

    Lu, Dong; Li, Wenjian; Wu, Xin; Wang, Jufang; Ma, Shuang; Liu, Qingfang; He, Jinyu; Jing, Xigang; Ding, Nan; Dai, Zhongying; Zhou, Jianping

    2010-12-01

    Yeast strain Saccharomyces cerevisiae was irradiated with different doses of 85 MeV/u 20Ne10+ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks (DSBs) and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T→G and T→C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.

  1. Programmable autonomous synthesis of single-stranded DNA

    Science.gov (United States)

    Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

    2018-02-01

    DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

  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. Role of DNA-PK in cellular responses to DNA double-strand breaks

    International Nuclear Information System (INIS)

    Chen, D.J.

    2003-01-01

    DNA double-strand breaks (DSBs) are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination. The repair of DSBs is of paramount importance to the cell as misrepair of DSBs can lead to cell death or promote tumorigenesis. In eukaryotes there exists two distinct mechanisms for DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, however, it is clear that nonhomologous repair of DSBs is highly active and plays a major role in conferring radiation resistance to the cell. The NHEJ machinery minimally consists of the DNA-dependent Protein Kinase (DNA-PK) and a complex of XRCC4 and DNA Ligase IV. The DNA-PK complex is composed of a 470 kDa catalytic subunit (DNA-PKcs), and the heterodimeric Ku70 and Ku80 DNA end-binding complex. DNA-PKcs is a PI-3 kinase with homology to ATM and ATR in its C-terminal kinase domain. The DNA-PK complex protects and tethers the ends, and directs assembly and, perhaps, the activation of other NHEJ proteins. We have previously demonstrated that the kinase activity of DNA-PK is essential for DNA DSB repair and V(D)J recombination. It is, therefore, of immense interest to determine the in vivo targets of DNA-PKcs and the mechanisms by which phosphorylation of these targets modulates NHEJ. Recent studies have resulted in the identification of a number of protein targets that are phosphorylated by and/or interact with DNA-PKcs. Our laboratory has recently identified autophosphorylation site(s) on DNA-PKcs. We find that phosphorylation at these sites in vivo is an early and essential response to DSBs and demonstrate, for the first time, the localization of DNA-PKcs to the sites of DNA damage in vivo. Furthermore, mutation of these phosphorylation sites in mammalian

  4. Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Filatov, M.V.; Noskin, L.A. (Leningrad Inst. of Nuclear Physics, Gatchina (USSR))

    1983-08-01

    Inhibitors of DNA synthesis 1-..beta..-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells' ability for excision repair. By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared. A scheme is proposed to describe molecular events associated with the phenomenon studied. 35 refs.

  5. DNA-repair, cell killing and normal tissue damage

    International Nuclear Information System (INIS)

    Dahm-Daphi, J.; Dikomey, E.; Brammer, I.

    1998-01-01

    Background: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. Material and methods: Review. Results: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. Conclusion: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly. (orig.) [de

  6. Role of DNA damage repair capacity in radiation induced adaptive response

    International Nuclear Information System (INIS)

    Yuan Dexiao; Pan Yan; Zhao Meijia; Chen Honghong; Shao Cunlin

    2009-01-01

    This work was to explore γ-ray induced radioadaptive response (RAR) in Chinese hamster ovary(CHO) cell lines of different DNA damage repair capacities. CHO-9 cells and the two repair-deficient strains, EM-C11(DNA single strand break repair deficient) and XR-C1(DNA double strand break repair deficient), were irradiated with a priming dose of 0.08 Gy or 0.016 Gy. After 4 or 7 hours, they were irradiated again with a challenging dose of 1 Gy. The micronucleus induction and plating efficiency of the cells were assayed. Under 0.08 Gy priming dose and 4-h interval, just the CHO-9 cells showed RAR, while with the 7-h interval the CHO-9 and EM-C11 showed RAR, but XR-C1 did not. When the cells were pretreated with a lower priming dose of 0.016 Gy in a 4-h time interval, all the three cell lines showed RAR to subsequent 1 Gy irradiation. It can be concluded that RAR is not only related to the priming dose and time interval, but also has close dependence on the ability of DNA damage repair. (authors)

  7. Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

    2002-01-01

    Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

  8. Iodine-125 induced DNA strand breakage: Contributions of different physical and chemical radiation action mechanisms

    International Nuclear Information System (INIS)

    Li, W.

    2002-01-01

    The decay of the radioisotope 125 I into 125 Te is typically followed by the emission of two groups of approximately 10 electrons each. In deoxyribonucleic acid (DNA) with 125 I incorporated, these electrons produce various types of damage to DNA, e.g. single and double strand breaks. They occur through direct actions of physical tracks, or indirect actions of radicals produced in water. Among the direct actions one should consider not only the excitation and ionization of DNA by electrons but also the neutralization of highly charged 125m Te ions with electrons from neighboring molecules. The present work begins with a detailed description of electron tracks with the use of the PARTRAC code, compares results with recent experiments, and concludes with a firm assessment of the contribution to the strand break yields from the neutralization effect. (orig.)

  9. Single Nucleotide Polymorphisms in Noncoding Regions of Rad51C Do Not Change the Risk of Unselected Breast Cancer but They Modulate the Level of Oxidative Stress and the DNA Damage Characteristics

    DEFF Research Database (Denmark)

    Gresner, Peter; Gromadzinska, Jolanta; Jablonska, Ewa

    2014-01-01

    affect the unselected BrC risk. Contrary to this, carriers of rs12946522, rs16943176, rs12946397 and rs17222691 rare-alleles were found to present significantly increased level of blood plasma TBARS compared to respective wild-type homozygotes (p... decreased fraction of oxidatively generated DNA damage (34% of total damaged DNA) in favor of DNA strand breakage, with no effect on total DNA damage, unlike respective wild-types, among which more evenly distributed proportions between oxidatively damaged DNA (48% of total DNA damage) and DNA strand...

  10. Spatiotemporal kinetics of γ-H2AX protein on charged particles induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Niu, H., E-mail: hniu@mx.nthu.edu.tw [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Chang, H.C. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China); Cho, I.C. [Institute for Radiological Research, Chang Gung University and Chang Gung Memorial Hospital, Taoyuan, Taiwan (China); Chen, C.H. [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, C.S. [Cancer Center of Taipei Veterans General Hospital, Taipei, Taiwan (China); Chou, W.T. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China)

    2014-08-15

    Highlights: • Charged particles can induce more complex DNA damages, and these complex damages have higher ability to cause the cell death or cell carcinogenesis. • In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in particle irradiated HeLa cells. • The HeLa cells were irradiated by 400 keV alpha-particles in four different dosages. • The result shows that a good linear relationship can be observed between foci number and radiation dose. • The data shows that the dissolution rate of γ-H2AX foci agree with the two components DNA repairing model, and it was decreasing as the radiation dose increased. • These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA repair. - Abstract: In several researches, it has been demonstrated that charged particles can induce more complex DNA damages. These complex damages have higher ability to cause the cell death or cell carcinogenesis. For this reason, clarifying the DNA repair mechanism after charged particle irradiation plays an important role in the development of charged particle therapy and space exploration. Unfortunately, the detail spatiotemporal kinetic of DNA damage repair is still unclear. In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in alpha-particle irradiated HeLa cells. The result shows that the intensity of γ-H2AX foci increased gradually, and reached to its maximum at 30 min after irradiation. A good linear relationship can be observed between foci intensity and radiation dose. After 30 min, the γ-H2AX foci intensity was decreased with time passed, but remained a large portion (∼50%) at 48 h passed. The data show that the dissolution rate of γ-H2AX foci agreed with two components DNA repairing model. These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA

  11. Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation

    Energy Technology Data Exchange (ETDEWEB)

    Asaithamby, Aroumougame, E-mail: Aroumougame.Asaithamy@UTsouthwestern.edu [Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 (United States); Chen, David J., E-mail: David.Chen@UTsouthwestern.edu [Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 (United States)

    2011-06-03

    Low-linear energy transfer (LET) radiation (i.e., {gamma}- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles is slowly repaired or is irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure.

  12. Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation

    International Nuclear Information System (INIS)

    Asaithamby, Aroumougame; Chen, David J.

    2011-01-01

    Low-linear energy transfer (LET) radiation (i.e., γ- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles is slowly repaired or is irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure.

  13. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    Science.gov (United States)

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. 75 FR 62820 - Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA

    Science.gov (United States)

    2010-10-13

    ... Providers of Synthetic Double- Stranded DNA AGENCY: Department of Health and Human Services, Office of the.... Government has developed Guidance that provides a framework for screening synthetic double-stranded DNA (dsDNA). This document, the Screening Framework Guidance for Providers of Synthetic Double-Stranded DNA...

  15. 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)

  16. Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

    Science.gov (United States)

    Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

    2014-10-01

    DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-23

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

  18. Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

    Science.gov (United States)

    Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

    2015-05-22

    HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

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

    2018-01-09

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

  20. Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

    Science.gov (United States)

    Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

    2014-06-17

    Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

  1. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    Science.gov (United States)

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

  2. Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells.

    Science.gov (United States)

    Huh, Yang Hoon; Cohen, Justin; Sherley, James L

    2013-10-15

    Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

  3. Flavonoids with DNA strand-scission activity from Rhus javanica var. roxburghiana.

    Science.gov (United States)

    Lin, Chun-Nan; Chen, Hui-Ling; Yen, Ming-Hong

    2008-01-01

    The flavonoids isolated from the stems of Rhus javanica var. roxburghiana, taxifolin (1), fisetin (2), fustin (3), 3,7,4'-trihydroxyflavanone (4) and 3,7,4'-trihydroxyflavone (5) caused breakage of supercoiled plasmid pBR322 DNA in the presence of Cu(II). Cu(I) was shown to be an essential intermediate by using the Cu(I)-specific sequestering reagent neocuproine. The Cu(II)-mediated DNA scissions induced by 1, 2, 3 and 5 were inhibited by the addition of catalase and exhibited DNA strand break by the addition of KI and superoxide dimutase (SOD), while in the Cu(II)-mediated DNA scissions induced by 4 was inhibited by the addition of KI, SOD, and catalase. It is concluded that 1, 2, 3, and 5 can induce H2O2 and superoxide anion, while 4 can induce OH* and H2O2 and subsequent oxidative damage of DNA in the presence of Cu(II).

  4. Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

    Science.gov (United States)

    Gan, Haiyun; Yu, Chuanhe; Devbhandari, Sujan; Sharma, Sushma; Han, Junhong; Chabes, Andrei; Remus, Dirk; Zhang, Zhiguo

    2017-10-19

    The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Initial events in the cellular effects of ionizing radiations: clustered damage in DNA

    International Nuclear Information System (INIS)

    Goodhead, D.T.

    1994-01-01

    Ionizing radiations produce many hundreds of different simple chemical products in DNA and also multitudes of possible clustered combinations. The simple products, including single-strand breaks, tend to correlate poorly with biological effectiveness. Even for initial double-strand breaks, as a broad class, there is apparently little or no increase in yield with increasing ionization density, in contrast with the large rise in relative biological effectiveness for cellular effects. Track structure analysis has revealed that clustered DNA damage of severity greater than simple double-strand breaks is likely to occur at biologically relevant frequencies with all ionizing radiations. Studies are in progress to describe in more detail the chemical nature of these clustered lesions and to consider the implications for cellular repair. (author)

  6. The use of recombinant DNA techniques to study radiation-induced damage, repair and genetic change in mammalian cells

    International Nuclear Information System (INIS)

    Thacker, J.

    1986-01-01

    A brief introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail. Examples are included of studies with both 254 nm ultraviolet light and ionizing radiation and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses. The review is dealt with under the following headings: Molecular distribution of DNA damage, The use of DNA-mediated gene transfer to assess damage and repair, The DNA double strand break: use of restriction endonucleases to model radiation damage, Identification and cloning of DNA repair genes, Analysis of radiation-induced genetic change. (UK)

  7. Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

    Directory of Open Access Journals (Sweden)

    Elise Darmon

    Full Text Available DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

  8. Assessment of DNA damage in radiation workers by using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Jia Lili; Zhang Tao; Yang Yonghua; Wang Yan; Du Liqing; Cao Jia; Wang Hong; Liu Qiang; Fan Feiyue

    2010-01-01

    Objective: To assess the DNA damage of radiation workers in different grade hospitals, and to explore the correlation between the types of work or work time and the levels of DNA damage. Methods: DNA single strand break were detected by using alkaline single cell gel electrophoresis (SCGE), and the comet was analyzed with CASP (Comet Assay Software Project). TDNA%, TL, TM and OTM were calculated. Results: The parameters of SCGE in the radiation group were higher than those of control group (F=3.93, P<0.01). The significant difference was found not only among the different types of work or different work time, but also among the different grade hospitals (F=1.83, 1.91, P<0.05). Conclusions: Various levels of DNA damage could be detected in the radiation workers of the two hospitals. DNA damage of radiation workers is less serious in the higher-grade hospital than the lower grade one. Different types of work or work time might affect the DNA damage level. (authors)

  9. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

    Science.gov (United States)

    Stengel, Gudrun; Kuchta, Robert D

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  10. Influence of DNA conformation on radiation-induced single-strand breaks

    International Nuclear Information System (INIS)

    Barone, F.; Belli, M.; Mazzei, F.

    1994-01-01

    We performed experiments on two DNA fragments of about 300 bp having different conformation to test whether radiation-induced single-strand breakage is dependent on DNA conformation. Breakage analysis was carried out by denaturing polyacrylamide gel electrophoresis, which allows determination of the broken site at single nucleotide resolution. We found uniform cutting patterns in B-form regions. On the contrary, X- or γ-irradiation of curved fragments of kinetoplast DNA showed that the distribution of single-strand breaks was not uniform along the fragment, as the cleavage pattern was modulated in phase with the runs of A-T pairs. This modulation likely reflected the reduced accessibility of the sites which on hydroxyl-radical attack give rise to strand breaks. The cleavage pattern was phased with the runs of A-T pairs. Moreover, the overall yield of strand breaks was considerably lower in curved DNA fragments than in those with extended straight regions. The conformation effect found here indicates that the cleavage pattern reflects the fine structural features of DNA. (orig./MG)

  11. Monte Carlo simulation of ionizing radiation induced DNA strand breaks utilizing coarse grained high-order chromatin structures.

    Science.gov (United States)

    Liang, Ying; Yang, Gen; Liu, Feng; Wang, Yugang

    2016-01-07

    Ionizing radiation threatens genome integrity by causing DNA damage. Monte Carlo simulation of the interaction of a radiation track structure with DNA provides a powerful tool for investigating the mechanisms of the biological effects. However, the more or less oversimplification of the indirect effect and the inadequate consideration of high-order chromatin structures in current models usually results in discrepancies between simulations and experiments, which undermine the predictive role of the models. Here we present a biophysical model taking into consideration factors that influence indirect effect to simulate radiation-induced DNA strand breaks in eukaryotic cells with high-order chromatin structures. The calculated yields of single-strand breaks and double-strand breaks (DSBs) for photons are in good agreement with the experimental measurements. The calculated yields of DSB for protons and α particles are consistent with simulations by the PARTRAC code, whereas an overestimation is seen compared with the experimental results. The simulated fragment size distributions for (60)Co γ irradiation and α particle irradiation are compared with the measurements accordingly. The excellent agreement with (60)Co irradiation validates our model in simulating photon irradiation. The general agreement found in α particle irradiation encourages model applicability in the high linear energy transfer range. Moreover, we demonstrate the importance of chromatin high-order structures in shaping the spectrum of initial damage.

  12. Monte Carlo simulation of ionizing radiation induced DNA strand breaks utilizing coarse grained high-order chromatin structures

    International Nuclear Information System (INIS)

    Liang, Ying; Yang, Gen; Liu, Feng; Wang, Yugang

    2016-01-01

    Ionizing radiation threatens genome integrity by causing DNA damage. Monte Carlo simulation of the interaction of a radiation track structure with DNA provides a powerful tool for investigating the mechanisms of the biological effects. However, the more or less oversimplification of the indirect effect and the inadequate consideration of high-order chromatin structures in current models usually results in discrepancies between simulations and experiments, which undermine the predictive role of the models. Here we present a biophysical model taking into consideration factors that influence indirect effect to simulate radiation-induced DNA strand breaks in eukaryotic cells with high-order chromatin structures. The calculated yields of single-strand breaks and double-strand breaks (DSBs) for photons are in good agreement with the experimental measurements. The calculated yields of DSB for protons and α particles are consistent with simulations by the PARTRAC code, whereas an overestimation is seen compared with the experimental results. The simulated fragment size distributions for 60 Co γ irradiation and α particle irradiation are compared with the measurements accordingly. The excellent agreement with 60 Co irradiation validates our model in simulating photon irradiation. The general agreement found in α particle irradiation encourages model applicability in the high linear energy transfer range. Moreover, we demonstrate the importance of chromatin high-order structures in shaping the spectrum of initial damage. (paper)

  13. Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

    Science.gov (United States)

    Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

    2015-11-25

    DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

  14. Induction and repair of DNA base damage studied in X-irradiated CHO cells using the M. luteus extract

    International Nuclear Information System (INIS)

    Foehe, C.; Dikomey, E.

    1994-01-01

    DNA base damage was measured in Chinese hamster ovary cells X-irradiated under aerobic conditions using an extract of the bacterium Micrococcus luteus. The glycosylases and endonucleases present in this extract recognize damaged bases and convert them into strand breaks (termed endonuclease-sensitive sites, enss). Strand breaks were detected by the alkaline unwinding technique. The induction of enss was measured for X-ray doses ranging up to 45 Gy. The relative frequency of all enss related to all radiation induced strand breaks was 1.7 ± 0.4. Repair of enss was studied for a radiation dose of 45 Gy. The number of enss was found to decrease exponentially with time after irradiation with a half-time of τ enss = 37 ± 8 min. The repair kinetics that were also measured for all X-ray-induced DNA strand breaks were found to consist of three phases: fast, intermediate and slow. The intermediate phase was fitted under the assumption that this phase results from the information and repair of secondary single-strand breaks generated by enzymatic incision at the sites of base damage repair. (author)

  15. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    International Nuclear Information System (INIS)

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-01-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate

  16. Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

    Science.gov (United States)

    Rackwitz, Jenny; Bald, Ilko

    2018-03-26

    During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. 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.

  18. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    Science.gov (United States)

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  19. Global chromatin fibre compaction in response to DNA damage

    International Nuclear Information System (INIS)

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

    2011-01-01

    Highlights: ► Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. ► DNA repair foci are found in soluble chromatin. ► Biophysical analysis reveals global chromatin fibre compaction after DNA damage. ► DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to

  20. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny

    OpenAIRE

    Leyla Vahidi Ferdousi; Pierre Rocheteau; Romain Chayot; Benjamin Montagne; Zayna Chaker; Patricia Flamant; Shahragim Tajbakhsh; Miria Ricchetti

    2014-01-01

    International audience; The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their...

  1. Radiation and DNA

    Energy Technology Data Exchange (ETDEWEB)

    Riabchenko, N I

    1979-01-01

    Consideration is given to the effects of ionizing radiation on the structure of DNA. Physical and chemical methods of determining radiation damage to the primary (polynucleotide chain and nitrogenous base) and secondary (helical) structure of DNA are discussed, and the effects of ionizing radiation on deoxyribonucleoprotein complexes are considered. The radiolysis of DNA in vitro and in bacterial and mammalian cells is examined and cellular mechanisms for the repair of radiation-damaged DNA are considered, taking into account single-strand and double-strand breaks, gamma-radiation damage and deoxyribonucleoprotein-membrane complex damage. Postradiation DNA degradation in bacteria and lymphatic cells is also discussed.

  2. 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 differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet...... assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA...

  3. Transformation frequency of γ irradiated plasmid DNA and the enzymatic double strand break formation by incubation in a protein extract of Escherichia coli

    International Nuclear Information System (INIS)

    Schulte-Frohlinde, D.; Mark, F.; Ventur, Y.

    1994-01-01

    It was found that incubation of γ-irradiated or DNaseI-treated plasmid DNA in a protein extract of Escherichia coli leads to enzyme-induced formation of double strand breaks (dsb) in competition with repair of precursors of these dsb. A survival curve of the plasmid DNA (as determined by transformation of E. coli) was calculated on the basis of enzyme-induced dsb as well as those produced by irradiation assuming that they are lethal. The calculated D O value was the same as that measured directly by transformation of irradiated plasmid DNA. Two models are presented that fit the experimental survival data as a function of dose. One is based on damage formation in the plasmid DNA including enzymatic conversion of single strand damage into dsb (U-model), the other is an enzymatic repair saturation model based on Michaelis-Menten kinetics. (Author)

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

    Science.gov (United States)

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

    2018-05-01

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

  5. Characterization of a parallel-stranded DNA hairpin

    International Nuclear Information System (INIS)

    Germann, M.W.; Vogel, H.J.; Pon, R.T.; van de Sande, J.H.

    1989-01-01

    Recently, the authors have shown that synthetic DNA containing homooligomeric A-T base pairs can form a parallel-stranded intramolecular hairpin structure. In the present study, they have employed NMR and optical spectroscopy to investigate the structure of the parallel-stranded (PS) DNA hairpin 3'-d(T) 8 C 4 (A) 8 -3' and the related antiparallel (APS) hair 5'-d(T) 8 C 4 (A) 8 -3'. The parallel orientation of the strands in the PS oligonucleotide is achieved by introducing a 5'-5' phosphodiester linkage in the hairpin loop. Ultraviolet spectroscopic and fluorescence data of drug binding are consistent with the formation of PS and APS structures, respectively, in these two hairpins. Vacuum circular dichroism measurements in combination with theoretical CD calculations indicate that the PS structure forms a right-handed helix. 31 P NMR measurements indicate that the conformation of the phosphodiester backbone of the PS structure is not drastically different from that of the APS control. The presence of slowly exchanging imino protons at 14 ppm and the observation of nuclear Overhauser enhancement between imino protons and the AH-2 protons demonstrate that similar base pairing and base stacking between T and A residues occur in both hairpins. On the basis of NOESY measurements, they find that the orientation of the bases is in the anti region and that the sugar puckering is in the 2'-endo range. The results indicate a B-like conformation for each of the strands in the stem part of the PS hairpin and reverse Watson-Crick base pairing between the T and A residues. These data are consistent with a previously calculated structure for parallel-stranded DNA

  6. Phosphorylation of human INO80 is involved in DNA damage tolerance

    International Nuclear Information System (INIS)

    Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

    2012-01-01

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

  7. 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.

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

    International Nuclear Information System (INIS)

    Lyckesvärd, Madeleine Nordén; Kapoor, Nirmal; Ingeson-Carlsson, Camilla; Carlsson, Therese; Karlsson, Jan-Olof; Postgård, Per; Himmelman, Jakob; Forssell-Aronsson, Eva; Hammarsten, Ola; Nilsson, Mikael

    2016-01-01

    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.

  9. 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].

  10. Biophysical characterization of the association of histones with single-stranded DNA.

    Science.gov (United States)

    Wang, Ying; van Merwyk, Luis; Tönsing, Katja; Walhorn, Volker; Anselmetti, Dario; Fernàndez-Busquets, Xavier

    2017-11-01

    Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. 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.)

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

    Science.gov (United States)

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

    2015-01-01

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

  13. DNA damage and mutagenesis of lambda phage induced by gamma-rays

    International Nuclear Information System (INIS)

    Bertram, Heidi

    1988-01-01

    Lambda phage DNA was gamma irradiated in aqueous solution and strand breakage determined. Twice as much minor structural damage per lethal hit was found in this DNA compared with DNA from irradiated phage suspensions. The in vitro irradiated DNA was repackaged into infectious particles. Induction of mutations in the cI or cII cistron was scored using SOS-induced host cells. In vitro prepared particles were found to have second-order kinetics for mutagenesis induced by gamma rays indicating two pre-mutational events were necessary to produce a mutation, but bacteria-free phage suspensions ('lys-phage') showed single hit kinetics for mutagenesis after irradiation. Increase in the mutation rate in the phage particles was mainly due to minor lesions, i.e. ssb, als and unidentified base damage. In lys-phage, mutagenesis might be enhanced by clustered DNA damage - configuration not existing in pack-phage. Loss of infectivity was analysed in comparison with structural damage. All lesions contributed to biological inactivation. Minor lesions were tolerated by lambda phage to a limited extent. Major lesions (e.g. dsb) contributed most to infectivity loss and were considered lethal events. (U.K.)

  14. Induction of double-strand breaks in DNA of prokaryotes and eukaryotes and their repair. 1. Application of elastoviscosimetry for studying double-strand breaks in DNA of Escherichia coli induced by. gamma. -irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bresler, S E; Noskin, L A; Suslov, A V [AN SSSR, Leningrad. Inst. Yadernoj Fiziki

    1980-11-01

    It is shown that the method of elastoviscosimetry gives a possibility to record the formation of DNA double-strand breaks in Escherichia coli cells induced by ..gamma.. irradiation at doses close to D/sub 37/. The dependence of changes of elastoviscosity parameter on the dose (tau/sub 0/) passes through the maximum. It is shown that the ascending section of this curve (at minimum ..gamma.. irradiation doses) characterizes the relaxation process of the superspiralised chromosome in nucleotide of the E. coli. This relaxation is observed due to ..gamma.. induced damages which are not double-strand breaks. By the maximum position one can judge on a dose yield of the first DNA double-strand break, the descending part of the dose curve describes the kinetics of accumulation of breaks with the dose increase. The analysis of the data obtained gives the possibility to come to the conclusion that when applying a usual technique of irradiation and lysis of cells not providing for special measures on inhibition of endo-and exonuclease activity in ..gamma.. irradiated cells, the dose yield of double-strand breaks noticeably increases (by 4.2 times). In the case of an essential, though incomplete, inhibition of nuclease activities in ..gamma.. irradiated cells the dose yield of breaks approximately corresponds to the dose curve of inactivation of these cells (D/sub 37/12.5+-3.0 krad, the first double-strand break -at 14.5+-2.4 krad).

  15. Induction of double-strand breaks in DNA of prokaryotes and eukaryotes and their repair. 1. Application of elastoviscosimetry for studying double-strand breaks in DNA of Escherichia coli induced by γ-irradiation

    International Nuclear Information System (INIS)

    Bresler, S.E.; Noskin, L.A.; Suslov, A.V.

    1980-01-01

    It is shown that the method of elastoviscosimetry gives a possibility to record the formation of DNA double-strand breaks in Escherichia coli cells induced by γ irradiation at doses close to D 37 . The dependence of changes of elastoviscosity parameter on the dose (tau 0 ) passes through the maximum. It is shown that the ascending section of this curve (at minimum γ irradiation doses) characterizes the relaxation process of the superspiralised chromosome in nucleotide of the E. coli. This relaxation is observed due to γ induced damages which are not double-strand breaks. By the maximum position one can judge on a dose yield of the first DNA double-strand break, the descending part of the dose curve describes the kinetics of accumulation of breaks with the dose increase. The analysis of the data obtained gives the possibility to come to the conclusion that when applying a usual technique of irradiation and lysis of cells not providing for special measures on inhibition of endo-and exonuclease activity in γ irradiated cells, the dose yield of double-strand breaks noticeably increases (by 4.2 times). In the case of an essential, though incomplete, inhibition of nuclease activities in γ irradiated cells the dose yield of breaks approximately corresponds to the dose curve of inactivation of these cells (D 37 12.5+-3.0 krad, the first double-strand break -at 14.5+-2.4 krad)

  16. Radiation damage to DNA in DNA-protein complexes.

    Science.gov (United States)

    Spotheim-Maurizot, M; Davídková, M

    2011-06-03

    The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals. 2011 Elsevier B.V. All rights reserved.

  17. Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

    Science.gov (United States)

    Bañuelos, C A; Banáth, J P; MacPhail, S H; Zhao, J; Eaves, C A; O'Connor, M D; Lansdorp, P M; Olive, P L

    2008-09-01

    Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

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

    Science.gov (United States)

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

    2011-06-24

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

  19. Radiation-induced DNA damage in tumors and normal tissues. III. Oxygen dependence of the formation of strand breaks and DNA-protein crosslinks

    International Nuclear Information System (INIS)

    Zhang, H.; Wallen, C.A.; Wheeler, K.T.; Joch, C.J.

    1995-01-01

    Results from several laboratories, including ours, have suggested that measurements of radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) may be used to estimate the hypoxic fraction or fractional hypoxic volume of tumors and normal tissues. This suggestion has been predicated on both published and nonpublished information that (1) the oxygen dependence of the formation of strand breaks in irradiated mammalian cells is similar to the oxygen dependence of radiation-produced cell killing, and (2) the oxygen dependence of the formation of DPCs in irradiated mammalian cells is the mirror image of the oxygen dependence of radiation-induced cell killing. However, the published studies that attempted to determine the relationship between the oxygen dependence of the formation of strand breaks and the radiation sensitivity of mammalian cells were not performed at 37 degrees C, the exact oxygen concentrations were not always known, and the results were conflicting. In addition, most of the data on the oxygen dependence of the formation of DPCs are unpublished. Consequently, we have undertaken a comprehensive investigation of one cell line, 9L/Ro rat brain tumor cells, to determine if the shape of the oxygen dependence curve and the K m value for radiation-induced strand breaks and DPCs were similar when 9L cells were irradiated under both ideal gas-liquid equilibrium conditions at 4 degrees C and nonideal gas-liquid equilibrium conditions at 37 degrees C. At 4 degrees C under ideal gas-liquid equilibrium conditions, the K m for the formation of strand breaks was approximately 0.0045 mM, and Km for radiation sensitivity was approximately 0.005mM. A similar comparison for the formation of DPCs at 4 degrees C could not be made, because the efficiency of the formation of DPC was much lower at 4 degrees C than at 37 degrees C. 30 refs., 3 figs

  20. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Science.gov (United States)

    Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  1. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Matthew L Hirsch

    Full Text Available Human embryonic stem cells (hESCs are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  2. Single-strand DNA molecule translocation through nanoelectrode gaps

    International Nuclear Information System (INIS)

    Zhao Xiongce; Payne, Christina M; Cummings, Peter T; Lee, James W

    2007-01-01

    Molecular dynamics simulations were performed to investigate the translocation of single-strand DNA through nanoscale electrode gaps under the action of a constant driving force. The application behind this theoretical study is a proposal to use nanoelectrodes as a screening gap as part of a rapid genomic sequencing device. Preliminary results from a series of simulations using various gap widths and driving forces suggest that the narrowest electrode gap that a single-strand DNA can pass is ∼1.5 nm. The minimum force required to initiate the translocation within nanoseconds is ∼0.3 nN. Simulations using DNA segments of various lengths indicate that the minimum initiation force is insensitive to the length of DNA. However, the average threading velocity of DNA varies appreciably from short to long DNA segments. We attribute such variation to the different nature of drag force experienced by the short and long DNA segments in the environment. It is found that DNA molecules deform significantly to fit in the shape of the nanogap during the translocation

  3. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    International Nuclear Information System (INIS)

    Fukumoto, Yasunori; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint

  4. Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

    DEFF Research Database (Denmark)

    1998-01-01

    RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

  5. Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii

    OpenAIRE

    Stroud, A. L.

    2012-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). ...

  6. Evolutionary implications of inversions that have caused intra-strand parity in DNA

    Directory of Open Access Journals (Sweden)

    Wei John

    2007-06-01

    Full Text Available Abstract Background Chargaff's rule of DNA base composition, stating that DNA comprises equal amounts of adenine and thymine (%A = %T and of guanine and cytosine (%C = %G, is well known because it was fundamental to the conception of the Watson-Crick model of DNA structure. His second parity rule stating that the base proportions of double-stranded DNA are also reflected in single-stranded DNA (%A = %T, %C = %G is more obscure, likely because its biological basis and significance are still unresolved. Within each strand, the symmetry of single nucleotide composition extends even further, being demonstrated in the balance of di-, tri-, and multi-nucleotides with their respective complementary oligonucleotides. Results Here, we propose that inversions are sufficient to account for the symmetry within each single-stranded DNA. Human mitochondrial DNA does not demonstrate such intra-strand parity, and we consider how its different functional drivers may relate to our theory. This concept is supported by the recent observation that inversions occur frequently. Conclusion Along with chromosomal duplications, inversions must have been shaping the architecture of genomes since the origin of life.

  7. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

    International Nuclear Information System (INIS)

    Biedermann, K.A.; Sun, J.R.; Giaccia, A.J.; Tosto, L.M.; Brown, J.M.

    1991-01-01

    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair

  8. C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

    Science.gov (United States)

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

    2009-10-30

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

  9. C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

    Science.gov (United States)

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

    2009-01-01

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

  10. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  12. Biological defense mechanisms against DNA double-strand break and their possible medical applications

    International Nuclear Information System (INIS)

    Matsumoto, Yoshihisa

    2011-01-01

    Radiation is now widely used for clinical diagnosis and therapeutics. On the other hand, radiation influences various tissues represented by immunological and reproductive systems, and is also recognized as one of the cause of carcinogenesis. Such pleiotropic effects of radiation are mediated through generation of damages on DNA molecule, vitally important genetic macromolecule. Among various types of DNA damages, double-strand break (DSB) is considered most critical and, therefore, responsible for biological effects. DSB is repaired mainly through two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Understanding of these mechanisms has been greatly deepened in past 20 years and is now providing a promising approach toward cancer therapy. We have studied the mechanisms of NHEJ, focusing especially on the role of phosphorylation and the assembly of machinery therein, which will be introduced below. (author)

  13. Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

    International Nuclear Information System (INIS)

    Pinar, Beatriz; Henríquez-Hernández, Luis Alberto; Lara, Pedro C; Bordon, Elisa; Rodriguez-Gallego, Carlos; Lloret, Marta; Nuñez, Maria Isabel; De Almodovar, Mariano Ruiz

    2010-01-01

    DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity

  14. Irreversible electron attachment--a key to DNA damage by solvated electrons in aqueous solution.

    Science.gov (United States)

    Westphal, K; Wiczk, J; Miloch, J; Kciuk, G; Bobrowski, K; Rak, J

    2015-11-07

    The TYT and TXT trimeric oligonucleotides, where X stands for a native nucleobase, T (thymine), C (cytosine), A (adenine), or G (guanine), and Y indicates a brominated analogue of the former, were irradiated with ionizing radiation generated by a (60)Co source in aqueous solutions containing Tris as a hydroxyl radical scavenger. In the past, these oligomers were bombarded with low energy electrons under an ultra-high vacuum and significant damage to TXT trimers was observed. However, in aqueous solution, hydrated electrons do not produce serious damage to TXT trimers although the employed radiation dose exceeded many times the doses used in radiotherapy. Thus, our studies demonstrate unequivocally that hydrated electrons, which are the major form of electrons generated during radiotherapy, are a negligible factor in damage to native DNA. It was also demonstrated that all the studied brominated nucleobases have a potential to sensitize DNA under hypoxic conditions. Strand breaks, abasic sites and the products of hydroxyl radical attachment to nucleobases have been identified by HPLC and LC-MS methods. Although all the bromonucleobases lead to DNA damage under the experimental conditions of the present work, bromopyrimidines seem to be the radiosensitizers of choice since they lead to more strand breaks than bromopurines.

  15. Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium.

    Science.gov (United States)

    Fern, Joshua; Schulman, Rebecca

    2017-09-15

    The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, in particular DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as the use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Together, these results provide a basic route to increased DNA circuit stability in cell culture environments.

  16. DNA damages induced by Ar F laser

    Energy Technology Data Exchange (ETDEWEB)

    Chapel, C.; Rose, S.; Chevrier, L.; Cordier, E.; Courant, D. [CEA Fontenay-aux-Roses, 92 (France). Dept. de Radiobiologie et de Radiopathologie

    2006-07-01

    The photo ablation process used in corneal refractive surgery by the Argon Fluoride (Ar F) laser emitting in ultraviolet C at 193 nm, exposes viable cells round the irradiated zone to sub ablative doses (< 400 joules.m -2). Despite that DNA absorption is higher at 193 nm than 254 nm, cytotoxicity of 193 nm laser radiation is lower than radiation emitted by 254 nm UV-C lamps. In situ, DNA could be protected of laser radiation by cellular components. Consequently, some authors consider that this radiation does not induce genotoxic effect whereas others suspect it to be mutagenic. These lasers are used for fifteen years but many questions remain concerning the long term effects on adjacent cells to irradiated area. The purpose of this study is to describe the effect of 193 nm laser radiation on DNA of stromal keratocytes which are responsible of the corneal structure. The 193 nm laser irradiation induces directly DNA breakage in keratocytes as it has been shown by the comet assay under alkaline conditions. Two hours post irradiation, damages caused by the highest exposure (150 J.m-2) are not repaired as it has been measured with the Olive Tail Moment (product of tail length and tail DNA content). They give partly evidence of induction of an apoptotic process in cells where DNA could be too damaged. In order to characterize specifically double strand breaks, a comparative analysis by immunofluorescence of the H2 Ax histone phosphorylation (H2 Ax) has been performed on irradiated keratocytes and unirradiated keratocytes. Results show a dose dependent increase of the number of H2 Ax positive cells. Consequences of unrepaired DNA lesions could be observed by the generation of micronuclei in cells. Results show again an increase of micronuclei in laser irradiated cells. Chromosomal aberrations have been pointed out by cytogenetic methods 30 mn after irradiation. These aberrations are dose dependent (from 10 to 150 J.m-2). The number of breakage decreases in the long run

  17. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

    International Nuclear Information System (INIS)

    Elsen, S.; Collin-Faure, V.; Gidrol, X.; Lemercier, C.

    2013-01-01

    Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design. (authors)

  18. Electron attachment to DNA single strands: gas phase and aqueous solution.

    Science.gov (United States)

    Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

    2007-01-01

    The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers

  19. Reduction of spontaneous somatic mutation frequency by a low-dose X irradiation of Drosophila larvae and possible involvement of DNA single-strand damage repair.

    Science.gov (United States)

    Koana, Takao; Takahashi, Takashi; Tsujimura, Hidenobu

    2012-03-01

    The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.

  20. The cellular environment in computer simulations of radiation-induced damage to DNA

    International Nuclear Information System (INIS)

    Moiseenko, V.V.; Waker, A.J.; Prestwich, W.V.

    1998-01-01

    Radiation-induced DNA single- and double-strand breaks were modeled for 660 keV photon radiation and scavenger capacity mimicking the cellular environment. Atomistic representation of DNA in B form with a first hydration shell was utilized to model direct and indirect damage. Monte Carlo generated electron tracks were used to model energy deposition in matter and to derive initial spatial distributions of species which appear in the medium following radiolysis. Diffusion of species was followed with time, and their reactions with DNA and each other were modeled in an encounter-controlled manner. Three methods to account for hydroxyl radical diffusion in a cellular environment were tested: assumed exponential survival, time-limited modeling and modeling of reactions between hydroxyl radicals and scavengers in an encounter-controlled manner. Although the method based on modeling scavenging in an encounter-controlled manner is more precise, it requires substantially more computer resources than either the exponential or time-limiting method. Scavenger concentrations of 0.5 and 0.15 M were considered using exponential and encounter-controlled methods with reaction rate set at 3 x 10 9 dm 3 mol -1 s -1 . Diffusion length and strand break yields, predicted by these two methods for the same scavenger molarity, were different by 20%-30%. The method based on limiting time of chemistry follow-up to 10 -9 s leads to DNA damage and radical diffusion estimates similar to 0.5 M scavenger concentration in the other two methods. The difference observed in predictions made by the methods considered could be tolerated in computer simulations of DNA damage. (orig.)

  1. The cellular environment in computer simulations of radiation-induced damage to DNA

    International Nuclear Information System (INIS)

    Moiseenko, V.V.; Hamm, R.N.; Waker, A.J.; Prestwich, W.V.

    1988-01-01

    Radiation-induced DNA single- and double-strand breaks were modeled for 660 keV photon radiation and scavenger capacity mimicking the cellular environment. Atomistic representation of DNA in B form with a first hydration shell was utilized to model direct and indirect damage. Monte Carlo generated electron tracks were used to model energy deposition in matter and to derive initial spatial distributions of species which appear in the medium following radiolysis. Diffusion of species was followed with time, and their reactions with DNA and each other were modeled in an encounter-controlled manner. Three methods to account for hydroxyl radical diffusion in cellular environment were tested: assumed exponential survival, time-limited modeling and modeling of reactions between hydroxyl radicals and scavengers in an encounter-controlled manner. Although the method based on modeling scavenging in an encounter-controlled manner is more precise, it requires substantially more computer resources than either the exponential or time-limiting method. Scavenger concentrations of 0.5 and 0.15 M were considered using exponential and encounter-controlled methods with reaction rate set at 3x10 9 dm 3 mol -1 s-1. Diffusion length and strand break yields, predicted by these two methods for the same scavenger molarity, were different by 20%-30%. The method based on limiting time of chemistry follow-up to 10 -9 s leads to DNA damage and radical diffusion estimates similar to 0.5 M scavenger concentration in the other two methods. The difference observed in predictions made by the methods considered could be tolerated in computer simulations of DNA damage. (author)

  2. 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.

  3. The Degree of Radiation-Induced DNA Strand Breaks Is Altered by Acute Sleep Deprivation and Psychological Stress and Is Associated with Cognitive Performance in Humans.

    Science.gov (United States)

    Moreno-Villanueva, Maria; von Scheven, Gudrun; Feiveson, Alan; Bürkle, Alexander; Wu, Honglu; Goel, Namni

    2018-03-27

    Sleep deprivation is associated with impaired immune responses, cancer, and morbidity and mortality, and can degrade cognitive performance, although individual differences exist in such responses. Sleep deprivation induces DNA strand breaks and DNA base oxidation in animals, and psychological stress is associated with increased DNA damage in humans. It remains unknown whether sleep deprivation or psychological stress in humans affects DNA damage response from environmental stressors, and whether these responses predict cognitive performance during sleep deprivation. Sixteen healthy adults (ages 29-52;mean age±SD, 36.4±7.1 years;7 women) participated in a 5-day experiment involving two 8 hour time-in-bed [TIB] baseline nights, followed by 39 hours total sleep deprivation (TSD), and two 8-10 hour TIB recovery nights. A modified Trier Social Stress Test was conducted on the day after TSD. Psychomotor Vigilance Tests measured behavioral attention. DNA damage was assessed in blood cells collected at 5 time points, and blood cells were irradiated ex-vivo. TSD, alone or in combination with psychological stress, did not induce significant increases in DNA damage. By contrast, radiation-induced DNA damage decreased significantly in response to TSD, but increased back to baseline when combined with psychological stress. Cognitively-vulnerable individuals had more radiation-induced DNA strand breaks before TSD, indicating their greater sensitivity to DNA damage from environmental stressors. Our results provide novel insights into the molecular consequences of sleep deprivation, psychological stress, and performance vulnerability. They are important for situations involving sleep loss, radiation exposure and cognitive deficits, including cancer therapy, environmental toxicology, and space medicine.

  4. Three methods to determine the yields of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Erzgraeber, G.; Lapidus, I.L.

    1985-01-01

    A possibility of determining the yield of DNA double-strand breaks in cells of the Chinese hamster (V79-4) by finding the amount of DNA released as a result of breaks and by determining the relative sedimentation velocity of DNA-membrane complexes affected by ionizing radiations with different physical characteristics is discussed. Results of the analysis are compared with the data obtained by a traditional method of sedimentation in the neutral sucrose density gradient. Comparative characterization of the methods is discussed. The yields of DNA double-strand breaks determined by the suggested independent methods are in good agreement, which opens possibilities of studying induction and repair of double-strand breaks by means of simpler and more reliable methods

  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. 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.

  7. Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

    Science.gov (United States)

    Yang, Zhiyu; Price, Nathan E.; Johnson, Kevin M.

    2017-01-01

    Abstract Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. PMID:28531327

  8. Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

    Science.gov (United States)

    Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

    2012-08-01

    RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

  9. Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

    Science.gov (United States)

    Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

    2013-01-01

    RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

  10. Repair of X-ray-induced single-strand breaks by a cell-free system

    International Nuclear Information System (INIS)

    Seki, Shuji; Ikeda, Shogo; Tsutui, Ken; Teraoka, Hirobumi

    1990-01-01

    Repair of X-ray-induced single-strand breaks of DNA was studied in vitro using an exonuclease purified from mouse ascites sarcoma (SR-C3H/He) cells. X-ray-dose-dependent unscheduled DNA synthesis was primed by the exonuclease. Repair of X-ray-induced single-strand breaks in pUC19 plasmid DNA was demonstrated by agarose gel electrophoresis after incubating the damaged DNA with the exonuclease, DNA polymerase (Klenow fragment of DNA polymerase I or DNA polymerase β purified from SR-C3H/He cells), four deoxynucleoside triphosphates, ATP and DNA ligase (T4 DNA ligase or DNA ligase I purified from calf thymus). The present results suggested that the exonuclease is involved in the initiation of repair of X-ray-induced single-strand breaks in removing 3' ends of X-ray-damaged DNA. (author)

  11. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

    Directory of Open Access Journals (Sweden)

    Michael Van Meter

    2016-09-01

    Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  12. DNA strand scission by the novel antitumor antibiotic leinamycin

    International Nuclear Information System (INIS)

    Hara, Mitsunobu; Saitoh, Yutaka; Nakano, Hirofumi

    1990-01-01

    Leinamycin is a recently discovered antitumor antibiotic with an unusual 1,3-dioxo-1,2-dithiolane structure. It preferentially inhibits the incorporation of [ 3 H]thymidine into the acid-insoluble fraction of Bacillus subtilis. In vitro, leinamycin causes single-strand cleavage of supercoiled double-helical pBR322 DNA in the presence of thiol cofactors. Scavengers of oxygen radical did not suppress the DNA-cleaving activity. Thiol-activated leinamycin binds calf thymus DNA at 4 degree C and thermal treatment of the leinamycin-DNA adduct released a chemically modified leinamycin from the complex. The lack of cytotoxicity and DNA-cleaving activity for S-deoxyleinamycin indicates that the 1,3-dioxo-1,2-dithiolane moiety is essential for the activity of leinamycin. Thus, the primary cellular target of leinamycin appears to be DNA. It binds DNA and causes single-strand break at low concentrations, which may account for the potent antitumor activity

  13. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    International Nuclear Information System (INIS)

    Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

    2007-01-01

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of γH2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si 3 N 4 window showed a homogenous Hsp70 expression pattern

  14. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Anja [Institute for Experimental Physics II, University of Leipzig (Germany) and Faculty of Biology, Pharmacy and Psychology, University of Leipzig (Germany)]. E-mail: afiedler@uni-leipzig.de; Reinert, Tilo [Institute for Experimental Physics II, University of Leipzig (Germany); Tanner, Judith [Clinic and Polyclinic for Radiation Oncology, University of Halle-Wittenberg (Germany); Butz, Tilman [Institute for Experimental Physics II, University of Leipzig (Germany)

    2007-07-15

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone {gamma}H2AX. Our concern was to test the feasibility of {gamma}H2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of {gamma}H2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si{sub 3}N{sub 4} window showed a homogenous Hsp70 expression pattern.

  15. Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

    Science.gov (United States)

    Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

    2013-04-01

    The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

  16. The studies of effects of 60Co γ-rays on DNA damage and repair in tumor cells

    International Nuclear Information System (INIS)

    Su Liaoyuan; Huang Hanxian; Cen Jiannong

    1997-06-01

    The effects of 60 Co γ-rays and its combination with hyperthermia on DNA strand breaks and their repair in L 5178y cells were studied using alkaline elution technique. When the cells were heated at 43 degree C for 30 min, there was obvious inhibition of repair of DNA damage caused by γ-irradiation. The hyperthermia before irradiation produced better inhibiting effect than that after irradiation. The effects of radiation on DNA strand breaks and its repair in HL-60 cells and HL-60 (VCR) cells were analyzed using technique of hydroxyapatite chromatography and the results sowed that the extent of DNA strand breaks in HL-60 cells and HL-60 (VCR) cells induced by irradiation was not markedly different, but the power of repair of strand breaks and the radioresistance of function of DNA synthesis in HL-60 (VCR) cells were higher than those in HL-60 cells. The difference was obvious. The results suggest that the heterogeneity of radiosensitivity of leukemia cells is correlated with its drug resistance. (10 refs., 3 tabs.)

  17. Repair and gamma radiation-induced single- and double-strand breaks in DNA of Escherichia coli

    International Nuclear Information System (INIS)

    Petrov, S.I.

    1981-01-01

    Studies in the kinetics of repair of γ-radiation-induced single- and double-strand breaks in DNA of E. coli cells showed that double-strand DNA breaks are rejoined by the following two ways. The first way is conditioned by repair of single-strand breaks and represents the repair of ''oblique'' double-strand breaks in DNA, whereas the second way is conditioned by functioning of the recombination mechanisms and, to all appearance, represents the repair of ''direct'' double-strand breaks in DNA

  18. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand.

    Science.gov (United States)

    Yasukawa, Takehiro; Reyes, Aurelio; Cluett, Tricia J; Yang, Ming-Yao; Bowmaker, Mark; Jacobs, Howard T; Holt, Ian J

    2006-11-15

    Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5' ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands to DNA starts at defined loci, the most prominent of which maps, in mammalian mtDNA, in the vicinity of the site known as the light-strand origin.

  19. Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage.

    Science.gov (United States)

    Denissova, Natalia G; Nasello, Cara M; Yeung, Percy L; Tischfield, Jay A; Brenneman, Mark A

    2012-01-01

    Resveratrol has elicited many provocative anticancer effects in laboratory animals and cultured cells, including reduced levels of oxidative DNA damage, inhibition of tumor initiation and progression and induction of apoptosis in tumor cells. Use of resveratrol as a cancer-preventive agent in humans will require that its anticancer effects not be accompanied by damage to normal tissue stem or progenitor cells. In mouse embryonic stem cells (mESC) or early mouse embryos exposed to ethanol, resveratrol has been shown to suppress apoptosis and promote survival. However, in cells exposed to genotoxic stress, survival may come at the expense of genome stability. To learn whether resveratrol can protect stem cells from DNA damage and to study its effects on genomic integrity, we exposed mESC pretreated with resveratrol to ionizing radiation (IR). Forty-eight hours pretreatment with a comparatively low concentration of resveratrol (10 μM) improved survival of mESC >2-fold after exposure to 5 Gy of X-rays. Cells pretreated with resveratrol sustained the same levels of reactive oxygen species and DNA strand breakage after IR as mock-treated controls, but repaired DNA damage more rapidly and resumed cell division sooner. Frequencies of IR-induced mutation at a chromosomal reporter locus were not increased in cells pretreated with resveratrol as compared with controls, indicating that resveratrol can improve viability in mESC after DNA damage without compromising genomic integrity.

  20. 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.

  1. ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

    Science.gov (United States)

    Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

    2017-12-07

    Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

    OpenAIRE

    Khodakov, Dmitriy A.; Khodakova, Anastasia S.; Huang, David M.; Linacre, Adrian; Ellis, Amanda V.

    2015-01-01

    Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within doubl...

  3. The thyroid hormone receptor β induces DNA damage and premature senescence.

    Science.gov (United States)

    Zambrano, Alberto; García-Carpizo, Verónica; Gallardo, María Esther; Villamuera, Raquel; Gómez-Ferrería, Maria Ana; Pascual, Angel; Buisine, Nicolas; Sachs, Laurent M; Garesse, Rafael; Aranda, Ana

    2014-01-06

    There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate-activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.

  4. Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

    Science.gov (United States)

    Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Wang, Yinsheng; Gates, Kent S

    2017-06-20

    Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Yields of clustered DNA damage induced by charged-particle radiations of similar kinetic energy per nucleon: LET dependence in different DNA microenvironments

    International Nuclear Information System (INIS)

    Keszenman, D.J.; Sutherland, B.M.

    2010-01-01

    To determine the linear energy transfer (LET) dependence of the biological effects of densely ionizing radiation in relation to changes in the ionization density along the track, we measured the yields and spectrum of clustered DNA damages induced by charged particles of different atomic number but similar kinetic energy per nucleon in different DNA microenvironments. Yeast DNA embedded in agarose in solutions of different free radical scavenging capacity was irradiated with 1 GeV protons, 1 GeV/nucleon oxygen ions, 980 MeV/nucleon titanium ions or 968 MeV/nucleon iron ions. The frequencies of double-strand breaks (DSBs), abasic sites and oxypurine clusters were quantified. The total DNA damage yields per absorbed dose induced in non-radioquenching solution decreased with LET, with minor variations in radioquenching conditions being detected. However, the total damage yields per particle fluence increased with LET in both conditions, indicating a higher efficiency per particle to induce clustered DNA damages. The yields of DSBs and non-DSB clusters as well as the damage spectra varied with LET and DNA milieu, suggesting the involvement of more than one mechanism in the formation of the different types of clustered damages.

  6. A miR-590/Acvr2a/Rad51b Axis Regulates DNA Damage Repair during mESC Proliferation

    Directory of Open Access Journals (Sweden)

    Qidong Liu

    2014-12-01

    Full Text Available Embryonic stem cells (ESCs enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF, which maintains the self-renewal capability of mouse ESCs (mESCs, significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB and double-strand break (DSB damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.

  7. 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)

  8. Genetic polymorphisms of DNA double-strand break repair pathway genes and glioma susceptibility

    International Nuclear Information System (INIS)

    Zhao, Peng; Zou, Peng; Zhao, Lin; Yan, Wei; Kang, Chunsheng; Jiang, Tao; You, Yongping

    2013-01-01

    Genetic variations in DNA double-strand break repair genes can influence the ability of a cell to repair damaged DNA and alter an individual’s susceptibility to cancer. We studied whether polymorphisms in DNA double-strand break repair genes are associated with an increased risk of glioma development. We genotyped 10 potentially functional single nucleotide polymorphisms (SNPs) in 7 DNA double-strand break repair pathway genes (XRCC3, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM) in a case–control study including 384 glioma patients and 384 cancer-free controls in a Chinese Han population. Genotypes were determined using the OpenArray platform. In the single-locus analysis there was a significant association between gliomas and the LIG4 rs1805388 (Ex2 +54C>T, Thr9Ile) TT genotype (adjusted OR, 3.27; 95% CI, 1.87-5.71), as well as the TC genotype (adjusted OR, 1.62; 95% CI, 1.20-2.18). We also found that the homozygous variant genotype (GG) of XRCC4 rs1805377 (IVS7-1A>G, splice-site) was associated with a significantly increased risk of gliomas (OR, 1.77; 95% CI, 1.12-2.80). Interestingly, we detected a significant additive and multiplicative interaction effect between the LIG4 rs1805388 and XRCC4 rs1805377 polymorphisms with an increasing risk of gliomas. When we stratified our analysis by smoking status, LIG4 rs1805388 was associated with an increased glioma risk among smokers. These results indicate for the first time that LIG4 rs1805388 and XRCC4 rs1805377, alone or in combination, are associated with a risk of gliomas

  9. Radiation induced strand breaks and time scale for repair of broken strands in superinfecting phage lambda DNA in Escherichia coli lysogenic for lambda

    International Nuclear Information System (INIS)

    Johansen, I.; Boye, E.; Brustad, T.

    1975-01-01

    The production of the first radiation induced break in covalent lambda DNA molecules in pol + and pol A 1 lysogenic host cells was measured after exposure to electrons from a linear accelerator and transfer to alkaline detergent within 100 ms from the onset of irradiation. The results revealed the presence of an oxygen effect in DNA strand breakage. In both pol + and pol A 1 host cells the rate of production in nitrogen was 1.2x10 -12 DNA single strand breaks per rad per dalton as compared to 5x10 -12 in oxygen. The yields of strand breaks in lambda DNA in pol + host cells under oxygenated or anoxic conditions are independent of whether the cells are irradiated in buffer at room temperature, in buffer at ice temperature, or in growth medium at 37 0 C. These results indicate that enzymic repair of DNA strand breaks before analysis is insignificant in these experiments. The presence of an oxygen effect in DNA strand breakage under these conditions suggest that an actual difference exists between initial number of breaks produced in nitrogen and in oxygen. The kinetics of rejoining of broken molecules under optimal growth conditions was measured by incubating the irradiated host cells prior to lysis. In pol + host cells 50% of the lambda DNA molecules broken in presence of oxygen are rejoined within 10 to 20 seconds of incubation. A significantly lower recovery is seen in pol + host cells after irradiation in nitrogen. The rejoining of broken lambda DNA strands in pol A 1 host cells is impaired after irradiation in presence of oxygen as well as under anoxia. These results show that DNA polymerase I is needed for the rapid rejoining of radiation induced strand breaks in the DNA, and that oxygen promoted strand breaks are more easily rejoined than are those produced in nitrogen. (author)

  10. Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; López-Fernández, Carmen; Fernández, José Luis; Dávila-Rodríguez, Martha I.; Johnston, Stephen D.; Gosálvez, Jaime

    2014-01-01

    Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to

  11. Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells.

    Science.gov (United States)

    Holton, Nathaniel W; Andrews, Joel F; Gassman, Natalie R

    2017-09-05

    Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

  12. Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

    International Nuclear Information System (INIS)

    Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

    2003-01-01

    Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

  13. DNA strand breaks induced by electrons simulated with nanodosimetry Monte Carlo simulation code: NASIC

    International Nuclear Information System (INIS)

    Li, Junli; Qiu, Rui; Yan, Congchong; Xie, Wenzhang; Zeng, Zhi; Li, Chunyan; Wu, Zhen; Tung, Chuanjong

    2015-01-01

    The method of Monte Carlo simulation is a powerful tool to investigate the details of radiation biological damage at the molecular level. In this paper, a Monte Carlo code called NASIC (Nanodosimetry Monte Carlo Simulation Code) was developed. It includes physical module, pre-chemical module, chemical module, geometric module and DNA damage module. The physical module can simulate physical tracks of low-energy electrons in the liquid water event-by-event. More than one set of inelastic cross sections were calculated by applying the dielectric function method of Emfietzoglou's optical-data treatments, with different optical data sets and dispersion models. In the pre-chemical module, the ionised and excited water molecules undergo dissociation processes. In the chemical module, the produced radiolytic chemical species diffuse and react. In the geometric module, an atomic model of 46 chromatin fibres in a spherical nucleus of human lymphocyte was established. In the DNA damage module, the direct damages induced by the energy depositions of the electrons and the indirect damages induced by the radiolytic chemical species were calculated. The parameters should be adjusted to make the simulation results be agreed with the experimental results. In this paper, the influence study of the inelastic cross sections and vibrational excitation reaction on the parameters and the DNA strand break yields were studied. Further work of NASIC is underway (authors)

  14. Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Xie L

    2016-11-01

    Full Text Available Lisha Xie,1,* Tiancen Zhao,1,2,* Jing Cai,1 You Su,1 Zehua Wang,1 Weihong Dong1 1Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Obstetrics and Gynecology, Central Hospital of Wuhan, Wuhan, China *These authors contributed equally to this work Objective: The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX in human choriocarcinoma cells regarding DNA damage response. Methods: Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot. Results: Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63 but not in MTX-resistant cancer cells (A2780 and Hela after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53. Conclusion: The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma. Keywords: choriocarcinoma, chemotherapy hypersensitivity, DNA double-strand break, RAD51, p53

  15. DNA damage in human lymphocytes due to synergistic interaction between ionizing radiation and pesticide

    International Nuclear Information System (INIS)

    Kim, J. K.; Lee, K. H.; Lee, B. H.; Chun, K. J.

    2001-01-01

    Biological risks may arise from the possibility of the synergistic interaction between harmful factors such as ionizing radiation and pesticide. The effect of pesticide on radiation-induced DNA damage in human in human blood lymphocytes was evaluated by the single cell gel electrophoresis (SCGE) assay. The lymphocytes, with or without pretreatment of the pesticide, were exposed to 2.0 Gy of gamma ray. Significantly increased tail moment, which was a marker of DNA strand breaks in SCGE assay, showed an excellent dose-response relationship. The present study confirms that the pesticide has the cytotoxic effect on lymphocytes and that it interacts synergistically with ionizing radiationon DNA damage, as well

  16. Accumulation of DNA Double-Strand Breaks in Normal Tissues After Fractionated Irradiation

    International Nuclear Information System (INIS)

    Ruebe, Claudia E.; Fricke, Andreas; Wendorf, Juliane; Stuetzel, Annika; Kuehne, Martin; Ong, Mei Fang; Lipp, Peter; Ruebe, Christian

    2010-01-01

    Purpose: There is increasing evidence that genetic factors regulating the recognition and/or repair of DNA double-strand breaks (DSBs) are responsible for differences in radiosensitivity among patients. Genetically defined DSB repair capacities are supposed to determine patients' individual susceptibility to develop adverse normal tissue reactions after radiotherapy. In a preclinical murine model, we analyzed the impact of different DSB repair capacities on the cumulative DNA damage in normal tissues during the course of fractionated irradiation. Material and Methods: Different strains of mice with defined genetic backgrounds (SCID -/- homozygous, ATM -/- homozygous, ATM +/- heterozygous, and ATM +/+ wild-type mice) were subjected to single (2 Gy) or fractionated irradiation (5 x 2 Gy). By enumerating γH2AX foci, the formation and rejoining of DSBs were analyzed in organs representative of both early-responding (small intestine) and late-responding tissues (lung, kidney, and heart). Results: In repair-deficient SCID -/- and ATM -/- homozygous mice, large proportions of radiation-induced DSBs remained unrepaired after each fraction, leading to the pronounced accumulation of residual DNA damage after fractionated irradiation, similarly visible in early- and late-responding tissues. The slight DSB repair impairment of ATM +/- heterozygous mice was not detectable after single-dose irradiation but resulted in a significant increase in unrepaired DSBs during the fractionated irradiation scheme. Conclusions: Radiation-induced DSBs accumulate similarly in acute- and late-responding tissues during fractionated irradiation, whereas the whole extent of residual DNA damage depends decisively on the underlying genetically defined DSB repair capacity. Moreover, our data indicate that even minor impairments in DSB repair lead to exceeding DNA damage accumulation during fractionated irradiation and thus may have a significant impact on normal tissue responses in clinical

  17. Multiple repair pathways mediate cellular tolerance to resveratrol-induced DNA damage.

    Science.gov (United States)

    Liu, Ying; Wu, Xiaohua; Hu, Xiaoqing; Chen, Ziyuan; Liu, Hao; Takeda, Shunichi; Qing, Yong

    2017-08-01

    Resveratrol (RSV) has been reported to exert health benefits for the prevention and treatment of many diseases, including cancer. The anticancer mechanisms of RSV seem to be complex and may be associated with genotoxic potential. To better understand the genotoxic mechanisms, we used wild-type (WT) and a panel of isogenic DNA-repair deficient DT40 cell lines to identify the DNA damage effects and molecular mechanisms of cellular tolerance to RSV. Our results showed that RSV induced significant formation of γ-H2AX foci and chromosome aberrations (CAs) in WT cells, suggesting direct DNA damage effects. Comparing the survival of WT with isogenic DNA-repair deficient DT40 cell lines demonstrated that single strand break repair (SSBR) deficient cell lines of Parp1 -/- , base excision repair (BER) deficient cell lines of Polβ -/- , homologous recombination (HR) mutants of Brca1 -/- and Brca2 -/- and translesion DNA synthesis (TLS) mutants of Rev3 -/- and Rad18 -/- were more sensitive to RSV. The sensitivities of cells were associated with enhanced DNA damage comparing the accumulation of γ-H2AX foci and number of CAs of isogenic DNA-repair deficient DT40 cell lines with WT cells. These results clearly demonstrated that RSV-induced DNA damage in DT40 cells, and multiple repair pathways including BER, SSBR, HR and TLS, play critical roles in response to RSV- induced genotoxicity. Copyright © 2017. Published by Elsevier Ltd.

  18. Phototherapy causes DNA damage in peripheral mononuclear leukocytes in term infants.

    Science.gov (United States)

    Aycicek, Ali; Kocyigit, Abdurrahim; Erel, Ozcan; Senturk, Hakan

    2008-01-01

    Our aim was to determine whether endogenous mononuclear leukocyte DNA strand is a target of phototherapy. The study included 65 term infants aged between 3-10 days that had been exposed to intensive (n = 23) or conventional (n = 23) phototherapy for at least 48 hours due to neonatal jaundice, and a control group (n = 19). DNA damage was assayed by single-cell alkaline gel electrophoresis (comet assay). Plasma total antioxidant capacity and total oxidant status levels were also measured, and correlation between DNA damage and oxidative stress was investigated. Mean values of DNA damage scores in both the intensive and conventional phototherapy groups were significantly higher than those in the control group (p Total oxidant status levels in both the intensive and conventional phototherapy groups were significantly higher than those in the control group (p = 0.005). Mean (standard deviation) values were 18.1 (4.2), 16.9 (4.4), 13.5 (4.2) micromol H2O2 equivalent/L, respectively. Similarly, oxidative stress index levels in both the intensive and conventional phototherapy groups were significantly higher than those in the control group (p = 0.041). Plasma total antioxidant capacity and total bilirubin levels did not differ between the groups (p > 0.05). There were no significant correlations between DNA damage scores and bilirubin, total oxidant status and oxidative stress levels in either phototherapy group (p > 0.05). Both conventional phototherapy and intensive phototherapy cause endogenous mononuclear leukocyte DNA damage in jaundiced term infants.

  19. Measurement of spontaneous DNA damage and DNA repair capacity in healthy adult individuals from high and normal level natural radiation areas of Kerala, India

    International Nuclear Information System (INIS)

    Vivek Kumar, P.R.; Jaikrishan, G.; Das, Birajalaxmi

    2014-01-01

    Inhabitants of the south west coastal areas of Kerala receive high level natural radiation due to the presence of monazite ( 232 Th) in the beach sand. This provides a unique opportunity to investigate the biological effects of high level natural radiation on humans. This study evaluate basal DNA damage in 149 healthy adult male subjects (104 from high level natural radiation areas (HLNRA, > 1mSv year -1 ) and 45 from normal level natural radiation area (NLNRA, d''1mSv year -1 ) by the alkaline comet assay. Oxidative DNA damage (ENDO III, FPG and hOGG1-sensitive sites) was measured by the enzyme modified comet assay. Induction and rejoining of DNA strand breaks was measured after irradiating peripheral blood lymphocytes with 2 Gy or 4 Gy gamma radiation. Basal damage due to age and residential area of the donors showed significant interaction (P=0.001), when all subjects were analyzed using a general linear model (GLM). In subgroup analysis, basal damage increased with age in subjects from the NLNRA (P=0.007), while a significant negative correlation (P=0.01) was observed in subjects from HLNRA. Oxidative DNA damage was not influenced by age, smoking habit or residential area in the entire sample. Subjects of high dose group from HLNRA (>5.75 mSv/y, N=34) showed a significant reduction in the induction of DNA damage after 2 Gy (P=0.03) and 4 Gy (P=0.05) compared to subjects form NLNRA. The study showed increased rejoining of DNA strand breaks in subjects from HLNRA when measured at 7 minutes after irradiation (P=0.04). In this pilot study, a low basal damage in elderly subjects from HLNRA and a reduced induction of DNA damage after 2 Gy and 4 Gy irradiation in high dose group subjects from HLNRA might suggest a possible role of chronic low dose natural radiation on the induction of an in vivo radio adaptive response. However, our findings need more validation in a larger study population. (author)

  20. Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

    Science.gov (United States)

    Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2014-02-01

    Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

  1. The role of cytosine methylation on charge transport through a DNA strand

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu [Department of Electrical Engineering, University of Washington, Seattle, Washington 98195-2500 (United States); Govind, Niranjan, E-mail: niri.govind@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States)

    2015-09-07

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

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

    Science.gov (United States)

    Parsons, J L; Chipman, J K

    2000-07-01

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

  3. How Does Thymine DNA Survive Ultrafast Dimerization Damage?

    Directory of Open Access Journals (Sweden)

    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.

  4. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    Energy Technology Data Exchange (ETDEWEB)

    Swindall, Amanda F.; Stanley, Jennifer A. [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Yang, Eddy S., E-mail: eyang@uab.edu [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States); Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States)

    2013-07-26

    Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

  5. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    International Nuclear Information System (INIS)

    Swindall, Amanda F.; Stanley, Jennifer A.; Yang, Eddy S.

    2013-01-01

    Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation

  6. Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

    International Nuclear Information System (INIS)

    Wang, Tzuchien V.; Smith, K.C.

    1986-01-01

    The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed. (Auth.)

  7. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Juanjuan; Zhang, Yu [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Xu, Wentao, E-mail: xuwentaoboy@sina.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Luo, YunBo [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Hao, Junran [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Shen, Xiao Li [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Yang, Xuan [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Li, Xiaohong [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Huang, Kunlun, E-mail: hkl009@163.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China)

    2013-04-15

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by

  8. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    International Nuclear Information System (INIS)

    Zheng, Juanjuan; Zhang, Yu; Xu, Wentao; Luo, YunBo; Hao, Junran; Shen, Xiao Li; Yang, Xuan; Li, Xiaohong; Huang, Kunlun

    2013-01-01

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ m ). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by OTA in

  9. Relationship between internal dosimetry and DNA double strand breaks in lymphocytes after radionuclide therapy; Zusammenhang zwischen physikalischer Dosimetrie und DNA Doppelstrangbruechen in Lymphozyten nach Radionuklidtherapie

    Energy Technology Data Exchange (ETDEWEB)

    Eberlein, Uta

    2015-09-30

    In radionuclide therapy radiopharmaceuticals are administered mostly systemically. Primarily, beta-emitters are used because of their short range in tissue. As a result the radiopharmaceutical distributes within the human body and accumulates in organs and target structures. Thus, the body is irradiated internally, in contrast to external irradiation in radiotherapy. The pattern of the activity distribution within the human body is determined by the physical and chemical properties of the radiopharmaceutical. Furthermore, the amount of activity and its accumulation in organs or tissues is essential for the calculation of the absorbed dose which defines the energy deposited in the body by ionizing radiation. During internal or external irradiation, patients are exposed to ionizing radiation which does not only destroy the malignant cells but also damages healthy tissue and cells. This is mainly caused by direct and indirect interaction of the radiation with the DNA which damages the DNA structure. Most frequently, there are single strand breaks and base damages. DNA double strand breaks (DSBs) are rare; nevertheless, they are the most critical lesions for cells as repairing the damage is difficult. Unrepaired or misrepaired DNA could cause mutations, chromosomal aberrations or lead to cell death. The formation of a DNA DSB in nuclear chromatin results in the rapid phosphorylation of the histone H2 variant H2AX, then called gamma-H2AX. Furthermore, DSBs also recruit the damage sensor 53BP1 to the chromatin surrounding the DSBs, which leads to 53BP1 and gamma-H2AX co-localization in the chromatin surrounding a DSB. By immunofluorescence staining with gamma-H2AX and 53BP1 antibodies those biomarkers can be addressed by microscopically visible DNA damage protein foci, this is also known as the DNA damage focus assay. With progression of DSB repair, gamma-H2AX and 53BP1 foci disappear. It is assumed that one focus corresponds to one DSB. Therefore, the number of foci per

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

    OpenAIRE

    Sterpone, Silvia; Cozzi, Renata

    2010-01-01

    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 de...

  11. Integrating DNA strand-displacement circuitry with DNA tile self-assembly

    Science.gov (United States)

    Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

    2013-01-01

    DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

  12. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    DEFF Research Database (Denmark)

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie

    2014-01-01

    The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sec...

  13. Life forms employ different repair strategies of repair single- and double strand DNA breaks caused by different qualities of radiation: criticality of RecA mediated repair system

    International Nuclear Information System (INIS)

    Sharan, R.N.

    2013-01-01

    Different qualities of radiation, either through direct or indirect pathway, induce qualitative different spectrum of damages in DNA, which are also different in in vitro and in vivo systems. The single- and double strand breaks of DNA are of special interest as they lead to serious biological consequences. The implications of such damage to DNA and their processing by various inherent repair pathways together decide the fate of the living form

  14. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    Science.gov (United States)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  15. Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis

    International Nuclear Information System (INIS)

    Smeets, M.F.M.A.; Mooren, E.H.M.; Begg, A.C.

    1993-01-01

    Radiation-induced DNA damage induction and repair was measured in two human squamous carcinoma cell lines with differing radiosensitivities. Experiments were carried out with field inversion gel electrophoresis (FIGE), adapted to measure DNA double strand break (DSB) induction and repair in unlabelled cells. The sensitivity of the method was increased by introducing a hybridization membrane into the agarose gel. Damaged DNA accumulated on one spot on the membrane resulting in high local concentrations. This DNA was quantified using radioactively-labelled total human DNA as a probe. Radiosensitivity differences at physiological temperatures could not be explained by differences in either induction or repair of DNA damage as measured by pulsed field gel electrophoresis. (author)

  16. Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants.

    Science.gov (United States)

    Charbonnel, Cyril; Gallego, Maria E; White, Charles I

    2010-10-01

    Double-strand breakage (DSB) of DNA involves loss of information on the two strands of the DNA fibre and thus cannot be repaired by simple copying of the complementary strand which is possible with single-strand DNA damage. Homologous recombination (HR) can precisely repair DSB using another copy of the genome as template and non-homologous recombination (NHR) permits repair of DSB with little or no dependence on DNA sequence homology. In addition to the well-characterised Ku-dependent non-homologous end-joining (NHEJ) pathway, much recent attention has been focused on Ku-independent NHR. The complex interrelationships and regulation of NHR pathways remain poorly understood, even more so in the case of plants, and we present here an analysis of Ku-dependent and Ku-independent repair of DSB in Arabidopsis thaliana. We have characterised an Arabidopsis xrcc1 mutant and developed quantitative analysis of the kinetics of appearance and loss of γ-H2AX foci as a tool to measure DSB repair in dividing root tip cells of γ-irradiated plants in vivo. This approach has permitted determination of DSB repair kinetics in planta following a short pulse of γ-irradiation, establishing the existence of a Ku-independent, Xrcc1-dependent DSB repair pathway. Furthermore, our data show a role for Ku80 during the first minutes post-irradiation and that Xrcc1 also plays such a role, but only in the absence of Ku. The importance of Xrcc1 is, however, clearly visible at later times in the presence of Ku, showing that alternative end-joining plays an important role in DSB repair even in the presence of active NHEJ. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

  17. The influence of bromodeoxyuridine on the induction and repair of DNA double-strand breaks in glioblastoma cells

    International Nuclear Information System (INIS)

    Nusser, N.N.; Bartkowiak, D.; Roettinger, E.M.

    2002-01-01

    Aims: To examine the dose response of DNA damage and its modification by the radiosensitizer, 5-bromo-2'-deoxyuridine (BrdU). The sensitizing mechanism is analyzed with regard to its influence on the induction and repair of DNA double-strand breaks (DSBs). Material and Methods: Cells from three different human glioblastoma lines, A7, LH and U87MG, were X-irradiated with and without exposure to BrdU. DNA fragments were separated by field-inversion gel electrophoresis (FIGE) and quantified by fluorometry immediately and 24 h after irradiation. Results: In all cell lines, the dose response followed a linear-quadratic rather than a purely linear function. BrdU-treated cells exhibited a significantly higher amount of mobile DNA. In repair experiments with and without BrdU, the amount of mobile DNA fell close to control values within 24 h. Conclusions: The linear-quadratic model appropriately describes the X-ray induced fragmentation of DNA. BrdU sensitizing acts predominantly by increasing DNA fragility, and not by impairing damage repair. The amount of DSBs persistent after 24 h of repair is minimal, even after highly cytotoxic doses. However, it appears to depend on the extent of initial damage, causing sensitized cells to retain more DSBs than unsensitized cells. (orig.)

  18. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

    NARCIS (Netherlands)

    Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

    2009-01-01

    In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to

  19. Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhary, Pankaj; Marshall, Thomas I. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast (United Kingdom); Currell, Frederick J. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast (United Kingdom); Centre for Plasma Physics, School of Mathematics and Physics, Queen' s University Belfast, Belfast (United Kingdom); Kacperek, Andrzej [Douglas Cyclotron, Clatterbridge Cancer Centre, Bebbington, Wirral (United Kingdom); Schettino, Giuseppe, E-mail: giuseppe.schettino@npl.co.uk [National Physical Laboratory, Teddington (United Kingdom); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast (United Kingdom)

    2016-05-01

    Purpose: To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams. Methods and Materials: Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, and distal positions of SOBP and the entrance and peak position for the pristine beam. Results: A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci. Conclusions: The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions.

  20. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    International Nuclear Information System (INIS)

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M.

    1990-01-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts

  1. Clusters of DNA damage induced by ionizing radiation: Formation of short DNA fragments. I. Theoretical modeling

    International Nuclear Information System (INIS)

    Holley, W.R.; Chatterjee, A.

    1996-01-01

    We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber composed of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and δ rays due to knock-on collisions involving energy transfers > 100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of circ OH, circ H, e aq , etc.; circ OH attack on sugar molecules leading to strand breaks; circ OH attack on bases; direct ionization of the sugar molecules leading to strand breaks; direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 hp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the chromatin fibers in mammalian DNA. 27 refs., 7 figs

  2. Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

    International Nuclear Information System (INIS)

    Tomita, Masanori

    2010-01-01

    Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

  3. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    International Nuclear Information System (INIS)

    Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

    2009-01-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

  4. DNA damage produced by exposure of supercoiled plasmid DNA to high- and low-LET ionizing radiation: Effects of hydroxyl radical quenchers. DNA breakage, neutrons, OH radicals

    International Nuclear Information System (INIS)

    Peak, J.G.; Ito, T.; Peak, M.J.; Robb, F.T.

    1994-01-01

    A supercoiled plasmid of 7300 base pairs was isolated and exposed in an aqueous environment to 60 Co γ rays and JANUS 0.85 MeV fission-spectrum neutrons. Dose responses for the production of single-strand breaks (SSBs), double-strand breaks (DSBs) and alkali-labile sites (ALSs) were compared with computations made from the conversion of the supercoil to its relaxed and linear forms. The relative biological effectiveness (RBE) for production of SSBs and DSBs was similar to that previously measured in the cellular environment. The RBE for destruction of genetic transforming activity of M13 viral DNA followed that for DNA damage. This is in contrast to the situation for biological effects such as lethality, mutagenesis, and cellular transformation measured in mammalian cells, where the RBE values are reversed. The role of hydroxyl (OH) radical in DNA damage induction by neutrons was investigated by exposure of plasmid in the presence of known quenchers of this species. Of four quenchers tested, all were able to reduce the yields of both SSBs and DSBs. These findings are consistent with a model for SSB and DSB induction by high linear energy transfer that involves OH radical mediation

  5. MEIOB targets single-strand DNA and is necessary for meiotic recombination.

    Directory of Open Access Journals (Sweden)

    Benoit Souquet

    Full Text Available Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB. This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/- spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/- meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

  6. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

    Science.gov (United States)

    Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

    2006-02-01

    During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

  7. Molecular investigation of evaporation of biodroplets containing single-strand DNA on graphene surface.

    Science.gov (United States)

    Akbari, Fahimeh; Foroutan, Masumeh

    2018-02-14

    In this study, the water droplet behaviour of four different types of single-strand DNA with homogeneous base sequence on a graphene substrate during evaporation of the droplet was investigated using molecular dynamics (MD) simulation. The simulation results indicated that the evaporation depended on the DNA sequence. The observed changes can be divided into four parts: (i) vaporization mode, (ii) evaporation flux, (iii) mechanism of single-strand placement on the surface, and (iv) consideration of remaining single strands after evaporation. Our simulation observations indicated different evaporation modes for thymine biodroplets as compared to those for other biodroplets. The evaporation of the thymine biodroplets occurred with an increase in the contact angle, while that of the other biodroplets occur in a constant contact angle mode. Moreover, thymine biodroplets generate the lowest contact line compared to other single strands, and it is always placed far away from the centre of the droplets during evaporation. Investigating variations in the evaporation flux shows that thymine has the highest evaporation flux and guanine has the lowest. Moreover, during initial evaporation, the flux of evaporation increases at the triple point of the biodroplets containing thymine single strands, while it decreases in the other biodroplets. The following observation was obtained from the study of the placement of single strands on the substrate: guanine and thymine interacted slower than other single strands during evaporation with graphene, adenine single strand had a higher folding during evaporation, and guanine single strand showed the lowest end-to-end distance. The investigation of single-strand DNA after evaporation shows that adenine produces the most stable structure at the end of evaporation. In addition, cytosine is the most stretched single-strand DNA due to its lack of internal π-π stacking and hydrogen bonding. Therefore, cytosine single strand is more

  8. A Binary-Encounter-Bethe Approach to Simulate DNA Damage by the Direct Effect

    Science.gov (United States)

    Plante, Ianik; Cucinotta, Francis A.

    2013-01-01

    The DNA damage is of crucial importance in the understanding of the effects of ionizing radiation. The main mechanisms of DNA damage are 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 in this area, many questions on the formation of DNA damage remains. To refine existing DNA damage models, an approach based on the Binary-Encounter-Bethe (BEB) model was developed[1]. This model calculates differential cross sections for ionization of the molecular orbitals of the DNA bases, sugars and phosphates using the electron binding energy, the mean kinetic energy and the occupancy number of the orbital. This cross section has an analytic form which is quite convenient to use and allows the sampling of the energy loss occurring during an ionization event. To simulate the radiation track structure, the code RITRACKS developed at the NASA Johnson Space Center is used[2]. This code calculates all the energy deposition events and the formation of the radiolytic species by the ion and the secondary electrons as well. We have also developed a technique to use the integrated BEB cross section for the bases, sugar and phosphates in the radiation transport code RITRACKS. These techniques should allow the simulation of DNA damage by ionizing radiation, and understanding of the formation of double-strand breaks caused by clustered damage in different conditions.

  9. A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell

    OpenAIRE

    Hong, Zehui; Jiang, Jie; Lan, Li; Nakajima, Satoshi; Kanno, Shin-ichiro; Koseki, Haruhiko; Yasui, Akira

    2008-01-01

    DNA double-strand breaks (DSBs) represent the most toxic DNA damage arisen from endogenous and exogenous genotoxic stresses and are known to be repaired by either homologous recombination or nonhomologous end-joining processes. Although many proteins have been identified to participate in either of the processes, the whole processes still remain elusive. Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in gene silencing, cancer development and the maintenance of embry...

  10. Inhibition of DNA-double strand break repair by antimony compounds

    International Nuclear Information System (INIS)

    Takahashi, Sentaro; Sato, Hiroshi; Kubota, Yoshihisa; Utsumi, Hiroshi; Bedford, Joel S.; Okayasu, Ryuichi

    2002-01-01

    DNA double strand breaks (DSBs), induced by γ-irradiation in Chinese hamster ovary cells, were used to examine whether antimony compounds affect the repair of DNA damage. The cells were first incubated with antimony trichloride or antimony potassium tartrate (both Sb(III)) for 2 h, and then irradiated with γ-rays at a dose of 40 Gy. The DNA DSB was quantified with pulsed field gel electrophoresis immediately after irradiation (non-repair group) as well as at 30 min post-irradiation (repair group). The degree of repair inhibition was determined by the differences in the amount of DNA DSB between non-repair and repair groups. Both antimony compounds inhibited repair of DNA DSB in a dose dependent manner. In trichloride, 0.2 mM antimony significantly inhibited the rejoining of DSB, while 0.4 mM was necessary in potassium antimony tartrate. The mean lethal doses, D 0 , for the treatment with antimony trichloride and antimony potassium tartrate, were approximately 0.21 and 0.12 mM, respectively. This indicates that the repair inhibition by antimony trichloride occurred in the dose range near D 0 , but the antimony potassium tartrate inhibited the repair at doses where most cells lost their proliferating ability. This is the first report to indicate that antimony compounds may inhibit the repair of radiation-induced DNA DSB

  11. Influence of anoxic sensitizers on the radiation damage in biologically active DNA in aqueous solution

    International Nuclear Information System (INIS)

    Lafleur, M.V.M.; Loman, H.

    1982-01-01

    The competition between biologically active single-stranded phiX174 DNA and the anoxic radiosensitizers metronidazole, misonidazole, paranitroacetophenone or nifuroxime for OH radicals is studied. The results are compared with experiments in which the protection of the DNA by t-butanol is determined. Also the effects of the sensitizers on the chemical nature of the damage (immediate and potential break, immediate and potential base damage) is studied. It is found that in diluted aqueous solutions of DNA these radiosensitizers do not sensitize with respect to the biological inactivation. The only effect observed is a shift from potential to immediate breaks with misonidazole and also nifuroxime. (author)

  12. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer.

    Science.gov (United States)

    Li, Tuo; Chen, Zhijian J

    2018-05-07

    Detection of microbial DNA is an evolutionarily conserved mechanism that alerts the host immune system to mount a defense response to microbial infections. However, this detection mechanism also poses a challenge to the host as to how to distinguish foreign DNA from abundant self-DNA. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates the adaptor protein STING. However, cGAS can be activated by double-stranded DNA irrespective of the sequence, including self-DNA. Although how cGAS is normally kept inactive in cells is still not well understood, recent research has provided strong evidence that genomic DNA damage leads to cGAS activation to stimulate inflammatory responses. This review summarizes recent findings on how genomic instability and DNA damage trigger cGAS activation and how cGAS serves as a link from DNA damage to inflammation, cellular senescence, and cancer. © 2018 Li and Chen.

  13. Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.

    Science.gov (United States)

    Choi, Eui-Hwan; Yoon, Seobin; Hahn, Yoonsoo; Kim, Keun P

    2017-02-01

    Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

  14. The Assessment of Primary DNA Damage in Medical Personnel Occupationally Exposed to Ionizing Radiation

    International Nuclear Information System (INIS)

    Kopjar, N.; Garaj-Vrhovac, V.

    2003-01-01

    In physico-chemical interaction with cellular DNA ionizing radiation produces a variety of primary lesions, such as single-strand breaks (SSB), alkali-labile sites, double-strand breaks (DSB), DNA-DNA and DNA-protein crosslinks, and damage to purine and pyrimidine bases. The effects of low-level exposure to ionising radiation are of concern to large number of people, including workers receiving radiation exposure on the job. It is very important to estimate absorbed doses from individuals occupationally exposed to ionising radiation for carrying out radioprotection procedures and restrict the hazards to human health. A wide range of methods is presently used for the detection of early biological effects of DNA-damaging agents in environmental and occupational settings. Currently, unstable chromosomal aberrations in peripheral blood lymphocytes, in particularly dicentrics, are the most fully developed biological indicators of ionizing radiation exposure. This methodology usually complements data obtained by physical dosimetry. As a routine, it is used whenever the individual dosimeter shows an exposure to penetrating radiation above its limit of detection. One of the advantages of cytogenetic dosimetry is that this biological dosimeter can be assessed at any moment whereas physical dosimeters are not always present in the subject. During the last years, the single cell gel electrophoresis (SCGE) or comet assay has gained widespread acceptance for genotoxicity testing. In molecular epidemiology studies DNA damage evaluated by the comet assay is utilized as a biomarker of exposure. The comet assay permits the detection of primary DNA damage and the study of repair kinetics at the level of single cells. The aim of the present study was to assess and quantificate the levels of DNA damage in peripheral blood leukocytes of medical workers occupationally exposed to ionizing radiation and corresponding unexposed control subjects. As a sensitive biomarker of exposure the

  15. Caffeine potentiates or protects against radiation-induced DNA and chromosomal damage in human lymphocytes depending on temperature and concentration

    International Nuclear Information System (INIS)

    Stoilov, L.M.; Mullenders, L.H.F.; Natarajan, A.T.

    1994-01-01

    The effect of caffeine on radiation-induced chromosomal aberrations and DNA strand breaks in unstimulated human lymphocytes was investigated. When present prior to and during the radiation exposure, caffeine treatment was found to cause either potentiation or protection against induction of chromosomal aberrations depending on the concentration and temperature. When the nucleoid sedimentation technique was applied, enhancement or reduction of radiation-induced DNA strand breaks by caffeine was also found to be dependent on temperature and caffeine concentration. It is proposed that caffeine, in addition to its suspected ability to influence DNA repair, can also influence the induction of DNA damage, leading to alterations in the yield of chromosomal aberrations

  16. Caffeine potentiates or protects against radiation-induced DNA and chromosomal damage in human lymphocytes depending on temperature and concentration

    Energy Technology Data Exchange (ETDEWEB)

    Stoilov, L.M. (Department of Molecular Genetics, Institute of Genetics, Sofia (Bulgaria)); Mullenders, L.H.F.; Natarajan, A.T. (J.A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden (Netherlands))

    1994-12-01

    The effect of caffeine on radiation-induced chromosomal aberrations and DNA strand breaks in unstimulated human lymphocytes was investigated. When present prior to and during the radiation exposure, caffeine treatment was found to cause either potentiation or protection against induction of chromosomal aberrations depending on the concentration and temperature. When the nucleoid sedimentation technique was applied, enhancement or reduction of radiation-induced DNA strand breaks by caffeine was also found to be dependent on temperature and caffeine concentration. It is proposed that caffeine, in addition to its suspected ability to influence DNA repair, can also influence the induction of DNA damage, leading to alterations in the yield of chromosomal aberrations.

  17. Study in regularities in the formation of double stranded DNA breaks in irradiated rat thymocytes

    International Nuclear Information System (INIS)

    Ivannik, B.P.; ProskuryakoV, S.Ya.; Ryabchenko, N.I.

    1979-01-01

    Using low-gradient viscosimetry of neutral detergent nuclear lysates a study was made of postradiation changes in the molecular weight of double-stranded DNA of thymocytes. It was established that 375 eV are needed for one double-stranded break to appear, and a dose of 1 rad is required for 0.275 double-stranded break to occur at the site of DNA with m.w. 10 12 dalton. The repair of double-stranded breaks is only observed when rats are exposed to a dose of 500 R. It is assumed that the absence of repair of double-stranded DNA breaks and the presence of secondary postradiation degradation of DNA are responsible for thymocyte death

  18. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...

  19. Ultrasound-induced DNA damage and signal transductions indicated by gammaH2AX

    Science.gov (United States)

    Furusawa, Yukihiro; Fujiwara, Yoshisada; Zhao, Qing-Li; Hassan, Mariame Ali; Ogawa, Ryohei; Tabuchi, Yoshiaki; Takasaki, Ichiro; Takahashi, Akihisa; Ohnishi, Takeo; Kondo, Takashi

    2011-09-01

    Ultrasound (US) has been shown to induce cancer cell death via different forms including apoptosis. Here, we report the potential of low-intensity pulsed US (LIPUS) to induce genomic DNA damage and subsequent DNA damage response. Using the ionizing radiation-induced DNA double-strand breaks (DSBs) as the positive control, we were able to observe the induction of DSBs (as neutral comet tails) and the subsequent formation of gammaH2AX-positive foci (by immunofluorescence detection) in human leukemia cells following exposure to LIPUS. The LIPUS-induced DNA damage arose most likely from the mechanical, but not sonochemical, effect of cavitation, based on our observation that the suppression of inertial cavitation abrogated the gammH2AX foci formation, whereas scavenging of free radical formation (e.g., hydroxyl radical) had no protective effect on it. Treatment with the specific kinase inhibitor of ATM or DNA-PKcs, which can phosphorylate H2AX Ser139, revealed that US-induced gammaH2AX was inhibited more effectively by the DNA-PK inhibitor than ATM kinase inhibitor. Notably, these inhibitor effects were opposite to those with radiation-induced gammH2AX. In conclusion, we report, for the first time that US can induce DNA damage and the DNA damage response as indicated by gammaH2AX was triggered by the cavitational mechanical effects. Thus, it is expected that the data shown here may provide a better understanding of the cellular responses to US.

  20. Enzymatic quantification of strand breaks of DNA induced by vacuum-UV radiation

    International Nuclear Information System (INIS)

    Ito, Takashi

    1986-01-01

    Hind3 digested plasmid DNA dried on an aluminum plate was irradiated by vacuum-UV at 160 and 195 nm using a synchrotron irradiation system. A change induced in the DNA, presumably a single strand break, was quantified by the aid of the strand break-derived stimulation of poly(ADP-ribose) synthetase activity. The end group of strand breaks so induced was recognized by the enzyme as effectively as that by DNase 1 treatment, suggesting a nicking as the major lesion inflicted on the DNA. The fluence (UV) dependent stimulation of poly(ADP-ribose) synthetase activity was much higher upon 160 nm irradiation than upon 195 nm irradiation. (Auth.)

  1. Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

    International Nuclear Information System (INIS)

    Kashino, Genro; Prise, Kevin M.; Schettino, Giuseppe; Folkard, Melvyn; Vojnovic, Borivoj; Michael, Barry D.; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

    2004-01-01

    This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells

  2. On the linearity of the dose-effect relationship of DNA double strand breaks

    International Nuclear Information System (INIS)

    Chadwick, K.H.; Leenhouts, H.P.

    1994-01-01

    Most radiation biologists believe that DNA double-strand breaks are induced linearly with radiation dose for all types of radiation. Since 1985, with the advent of elution and gel electrophoresis techniques which permit the measurement of DNA double-strand breaks induced in mammalian cells at doses having radiobiological relevance, the true nature of the dose-effect relationship has been brought into some doubt. Many investigators measured curvilinear dose-effect relationships and a few found good correlations between the induction of the DNA double-strand breaks and cell survival. We approach the problem pragmatically by assuming that the induction of DNA double-strand breaks by 125 I Auger electron emitters incorporated into the DNA of the cells is a linear function of the number of 125 I decays, and by comparing the dose-effect relationship for sparsely ionizing radiation against this standard. The conclusion drawn that the curvilinear dose-effect relationships and the correlations with survival are real. (Author)

  3. A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands.

    Science.gov (United States)

    Johnson, Robert E; Klassen, Roland; Prakash, Louise; Prakash, Satya

    2015-07-16

    Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  5. Mechanisms for radiation damage in DNA. Final report, June 1, 1986--August 31, 1996

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1996-08-01

    Over the last 10 years significant advances have been made impacting the understanding of radiation damage to DNA. The principal objective of this work was the elucidation of the fundamental mechanisms of radiation damage to DNA through the direct and indirect effects. Recently the work concentrated on the direct effect of radiation damage on DNA. The objective was to elucidate the ultimate radiation chemical damage to DNA arising from the direct effect. In this effort the focus was on the application of three techniques. ESR spectroscopic measurement of initial radicals formed in DNA and its hydration layer at low temperatures. Ab initio molecular orbital calculations were employed to give highly accurate theoretical predictions of early events such as electron and hole localization sites which serve to test and to clarify the experimental observations. HPLC and GC-mass spectroscopic assays of DNA base products formation provide the ultimate chemical outcome of the initial radiation events. The bridge between the early ion radical species and the non-radical products is made in ESR studies which follow the chemistry of the early species as they react with water and or other DNA bases. The use of these techniques has resulted in a new and fundamental understanding of the radiation damage to DNA on a molecular scale. From this work, a working model for DNA damage from the initial ionization event to the eventual formation of molecular base damage products and strand breaks has been formulated. Results over the past several years which have led to the formulation of this model are described

  6. Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Wojewodzka, M., E-mail: marylaw@ichtj.waw.pl [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Gradzka, I.; Buraczewska, I.; Brzoska, K.; Sochanowicz, B. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Goncharova, R.; Kuzhir, T. [Institute of Genetics and Cytology, Belarussian National Academy of Sciences, Minsk (Belarus); Szumiel, I. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland)

    2009-12-01

    Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

  7. Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

    Directory of Open Access Journals (Sweden)

    Yanhao Lai

    Full Text Available Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA, an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER. We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA