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Sample records for regulate dna repair

  1. Regulation of DNA repair by parkin

    International Nuclear Information System (INIS)

    Kao, Shyan-Yuan

    2009-01-01

    Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.

  2. Nuclear translocation contributes to regulation of DNA excision repair activities

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Andersen, Sofie Dabros; Lützen, Anne

    2009-01-01

    for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import......DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA......, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M...

  3. DNA repair

    International Nuclear Information System (INIS)

    Van Zeeland, A.A.

    1984-01-01

    In this chapter a series of DNA repair pathways are discussed which are available to the cell to cope with the problem of DNA damaged by chemical or physical agents. In the case of microorganisms our knowledge about the precise mechanism of each DNA repair pathway and the regulation of it has been improved considerably when mutants deficient in these repair mechanisms became available. In the case of mammalian cells in culture, until recently there were very little repair deficient mutants available, because in almost all mammalian cells in culture at least the diploid number of chromosomes is present. Therefore the frequency of repair deficient mutants in such populations is very low. Nevertheless because replica plating techniques are improving some mutants from Chinese hamsters ovary cells and L5178Y mouse lymphoma cells are now available. In the case of human cells, cultures obtained from patients with certain genetic diseases are available. A number of cells appear to be sensitive to some chemical or physical mutagens. These include cells from patients suffering from xeroderma pigmentosum, Ataxia telangiectasia, Fanconi's anemia, Cockayne's syndrome. However, only in the case of xeroderma pigmentosum cells, has the sensitivity to ultraviolet light been clearly correlated with a deficiency in excision repair of pyrimidine dimers. Furthermore the work with strains obtained from biopsies from man is difficult because these cells generally have low cloning efficiencies and also have a limited lifespan in vitro. It is therefore very important that more repair deficient mutants will become available from established cell lines from human or animal origin

  4. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  5. DNA repair

    International Nuclear Information System (INIS)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

  6. Acetylation regulates WRN catalytic activities and affects base excision DNA repair

    DEFF Research Database (Denmark)

    Muftuoglu, Meltem; Kusumoto, Rika; Speina, Elzbieta

    2008-01-01

    The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone...... acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription....

  7. Dynamic regulation of cerebral DNA repair genes by psychological stress

    DEFF Research Database (Denmark)

    Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta

    2015-01-01

    Neuronal genotoxic insults from oxidative stress constitute a putative molecular link between stress and depression on the one hand, and cognitive dysfunction and dementia risk on the other. Oxidative modifications to DNA are repaired by specific enzymes; a process that plays a critical role...... restraint stress (6h/day) or daily handling (controls), and sacrificed after 1, 7 or 21 stress sessions. The mRNA expression of seven genes (Ogg1, Ape1, Ung1, Neil1, Xrcc1, Ercc1, Nudt1) involved in the repair of oxidatively damaged DNA was determined by quantitative real time polymerase chain reaction...

  8. Regulation of DNA Alkylation Damage Repair: Lessons and Therapeutic Opportunities.

    Science.gov (United States)

    Soll, Jennifer M; Sobol, Robert W; Mosammaparast, Nima

    2017-03-01

    Alkylation chemotherapy is one of the most widely used systemic therapies for cancer. While somewhat effective, clinical responses and toxicities of these agents are highly variable. A major contributing factor for this variability is the numerous distinct lesions that are created upon alkylation damage. These adducts activate multiple repair pathways. There is mounting evidence that the individual pathways function cooperatively, suggesting that coordinated regulation of alkylation repair is critical to prevent toxicity. Furthermore, some alkylating agents produce adducts that overlap with newly discovered methylation marks, making it difficult to distinguish between bona fide damaged bases and so-called 'epigenetic' adducts. Here, we discuss new efforts aimed at deciphering the mechanisms that regulate these repair pathways, emphasizing their implications for cancer chemotherapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  10. Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

    Science.gov (United States)

    Kannappan, Ramaswamy; Mattapally, Saidulu; Wagle, Pooja A; Zhang, Jianyi

    2018-05-18

    The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells. Exposure to low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) measured by RAD50 and MRE11 expression, Checkpoint kinase 2 activation and γH2A.X recruitment at DNA strand breaks in both the cell groups indicating silencing p53-TAD do not affect DDR mechanism upstream of p53. Following removal of Doxo p53-WT hiPS cells underwent DNA repair, corrected their damaged DNA and restored DNA integrity. Conversely, p53-TAD KO hiPS cells did not undergo complete DNA repair and failed to restore DNA integrity. More importantly continuous culture of p53-TAD KO hiPS cells underwent G2/M cell cycle arrest and expressed cellular senescent marker p16 INK4a . Our data clearly shows that silencing transactivation domain of p53 did not affect DDR but affected the DNA repair process implying the crucial role of p53 transactivation domain in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent senescence.

  11. Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex

    DEFF Research Database (Denmark)

    Géli, Vincent; Lisby, Michael

    2015-01-01

    and colleagues shows that also physiological threats to genome integrity such as DNA secondary structure-forming triplet repeat sequences relocalize to the NPC during DNA replication. Mutants that fail to reposition the triplet repeat locus to the NPC cause repeat instability. Here, we review the types of DNA...... lesions that relocalize to the NPC, the putative mechanisms of relocalization, and the types of recombinational repair that are stimulated by the NPC, and present a model for NPC-facilitated repair....

  12. DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.

    Science.gov (United States)

    Serrano, M A; Li, Z; Dangeti, M; Musich, P R; Patrick, S; Roginskaya, M; Cartwright, B; Zou, Y

    2013-05-09

    Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

  13. Histone Variant Regulates DNA Repair via Chromatin Condensation | Center for Cancer Research

    Science.gov (United States)

    Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have

  14. DNA Repair Systems

    Indian Academy of Sciences (India)

    DNA molecule which makes it ideal for storage and propagation of genetic information. ... of these errors are broadly referred to as DNA repair. DNA can ... changes occur in the human genome per day. ..... nails, frequent physical and mental.

  15. Control of radiation sensitivity of mammalian cells. Regulation of expression of DNA repair genes

    International Nuclear Information System (INIS)

    Yoshida, Kayo; Morita, Takashi

    2003-01-01

    This review describes authors' investigations concerning regulation of expression of DNA repair genes for the purpose of control of radiosensitivity of mammalian cells for cancer radiotherapy. One of their experiments concerns the enhancement of sensitivity to radiation and anti-tumor agents by suppressing the expression of mammalian Rad51 gene which playing a central role in recombination repair against DNA double-strand break, by RNA interference (RNAi). Described are the mode of action of RNAi, mechanism of suppression of Rad51 gene expression by it, enhancing effect in radiosensitivity, stable suppression and enhancement by hairpin RNA and its possible usefulness in cancer therapy. The other concerns the histone H2AX gene, which delivering the repair signal post phosphorylation in chromatin against the double-strand break. Experimental results of suppression of the histone H2AX gene by tet-off system, enhancement of radiosensitivity by the suppression and functional recovery by the gene transfer are described, and the radiosensitivity can be thus artificially controlled by tetracycline in authors' F9 2AX (tet/tet) cells. (N.I.)

  16. A decade of understanding spatio-temporal regulation of DNA repair by the nuclear architecture.

    Science.gov (United States)

    Saad, Hicham; Cobb, Jennifer A

    2016-10-01

    The nucleus is a hub for gene expression and is a highly organized entity. The nucleoplasm is heterogeneous, owing to the preferential localization of specific metabolic factors, which lead to the definition of nuclear compartments or bodies. The genome is organized into chromosome territories, as well as heterochromatin and euchromatin domains. Recent observations have indicated that nuclear organization is important for maintaining genomic stability. For example, nuclear organization has been implicated in stabilizing damaged DNA, repair-pathway choice, and in preventing chromosomal rearrangements. Over the past decade, several studies have revealed that dynamic changes in the nuclear architecture are important during double-strand break repair. Stemming from work in yeast, relocation of a damaged site prior to repair appears to be at least partially conserved in multicellular eukaryotes. In this review, we will discuss genome and nucleoplasm architecture, particularly the importance of the nuclear periphery in genome stability. We will also discuss how the site of relocation regulates repair-pathway choice.

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

    DEFF Research Database (Denmark)

    Mund, Andreas; Schubert, Tobias; Staege, Hannah

    2012-01-01

    -dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non...

  18. DNA repair genes

    International Nuclear Information System (INIS)

    Morimyo, Mitsuoki

    1995-01-01

    Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

  19. miR-24-mediated down-regulation of H2AX suppresses DNA repair in terminally differentiated blood cells

    Science.gov (United States)

    Lal, Ashish; Pan, Yunfeng; Navarro, Francisco; Dykxhoorn, Derek M.; Moreau, Lisa; Meire, Eti; Bentwich, Zvi; Lieberman, Judy; Chowdhury, Dipanjan

    2010-01-01

    Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but the molecular mechanism behind this down-regulation is unclear. Here we find that miR-24 is consistently up-regulated during post-mitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a key DSB repair protein that activates cell cycle checkpoint proteins and retains DSB repair factors at DSB foci. The H2AX 3’UTR contains conserved miR-24 binding sites regulated by miR-24. Both H2AX mRNA and protein are substantially reduced during hematopoietic cell terminal differentiation by miR-24 up-regulation both in in vitro differentiated cells and primary human blood cells. miR-24 suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs. Antagonizing miR-24 in differentiating cells protects them from DNA damage-induced cell death, while transfecting miR-24 mimics in dividing cells increases chromosomal breaks and unrepaired DNA damage and reduces viability in response to DNA damage. This DNA repair phenotype can be fully rescued by over-expressing miR-24-insensitive H2AX. Therefore, miR-24 up-regulation in post-replicative cells reduces H2AX and thereby renders them highly vulnerable to DNA damage. PMID:19377482

  20. DNA repair , cell repair and radiosensitivity

    International Nuclear Information System (INIS)

    Zhestyanikov, V.D.

    1983-01-01

    Data obtained in laboratory of radiation cytology and literature data testifying to a considerable role of DNA repair in cell sensitivity to radiation and chemical DNA-tropic agents have been considered. Data pointing to the probability of contribution of inducible repair of DNA into plant cells sensitivity to X-rays are obtained. Certain violations of DNA repair do not result in the increase of radiosensitivity. It is assumed that in the cases unknown mechanisms of DNA repair operate

  1. Different domains of P21Cip1/waf1 regulate DNA replication and DNA repair-associated processes after UV

    International Nuclear Information System (INIS)

    Soria, Gaston; Speroni, Juliana; Podhajcer, Osvaldo L.; Gottifredi, Vanesa; Prives, Carol

    2007-01-01

    Full text: Many genotoxic insults result in p21 up-regulation and p21-dependent cell cycle arrest but UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated to DNA repair. While the role of p21 in Nucleotide Excision Repair (NER) remains controversial, two recent reports explore its effect on Translesion DNA Synthesis (TLS), a process that avoids replication blockage during S phase. The first report shows that p21 degradation is required for efficient PCNA ubiquitination, a post transcriptional modification that is relevant for TLS. The second report demonstrates that p21 (-/-) cells have increased TLS-associated mutagenic rates. Herein we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK binding domain of p21 is required for cell cycle arrest in unstressed cells; neither the CDK- nor the PCNA-binding domains of p21 are able to block early and late steps of NER. Intriguingly, through its PCNA binding domain, p21 inhibited recruitment of the TLS-polymerase, polη to PCNA foci after UV. Moreover, this obstruction correlates with accumulation of γH2AX and increased apoptosis. Taking together, our data emphasizes the link between p21 turnover and efficient TLS. This might also suggest a potential effect of p21 on other activities of polζ, a DNA polymerase with central roles in other biological scenarios such as genetic conversion, homologous recombination and modulation of the cellular response to genotoxic agents [es

  2. 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...

  3. Use of Drosophila to study DNA repair

    International Nuclear Information System (INIS)

    Boyd, J.B.; Harris, P.V.; Sakaguchi, K.

    1988-01-01

    This paper discusses Drosophila, the premier metazoan organism for analyzing many fundamental features of eukaryotic gene regulation. The authors present adaptations of several approaches for studying DNA repair to an analysis of repair-defective mutants in Drosophila. A current understanding of Drosophila DNA repair is described

  4. DNA repair and cancer

    International Nuclear Information System (INIS)

    Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

    2012-01-01

    DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

  5. Celebrating DNA's Repair Crew.

    Science.gov (United States)

    Kunkel, Thomas A

    2015-12-03

    This year, the Nobel Prize in Chemistry has been awarded to Tomas Lindahl, Aziz Sancar, and Paul Modrich for their seminal studies of the mechanisms by which cells from bacteria to man repair DNA damage that is generated by normal cellular metabolism and stress from the environment. These studies beautifully illustrate the remarkable power of DNA repair to influence life from evolution through disease susceptibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Nickel induces transcriptional down-regulation of DNA repair pathways in tumorigenic and non-tumorigenic lung cells.

    Science.gov (United States)

    Scanlon, Susan E; Scanlon, Christine D; Hegan, Denise C; Sulkowski, Parker L; Glazer, Peter M

    2017-06-01

    The heavy metal nickel is a known carcinogen, and occupational exposure to nickel compounds has been implicated in human lung and nasal cancers. Unlike many other environmental carcinogens, however, nickel does not directly induce DNA mutagenesis, and the mechanism of nickel-related carcinogenesis remains incompletely understood. Cellular nickel exposure leads to signaling pathway activation, transcriptional changes and epigenetic remodeling, processes also impacted by hypoxia, which itself promotes tumor growth without causing direct DNA damage. One of the mechanisms by which hypoxia contributes to tumor growth is the generation of genomic instability via down-regulation of high-fidelity DNA repair pathways. Here, we find that nickel exposure similarly leads to down-regulation of DNA repair proteins involved in homology-dependent DNA double-strand break repair (HDR) and mismatch repair (MMR) in tumorigenic and non-tumorigenic human lung cells. Functionally, nickel induces a defect in HDR capacity, as determined by plasmid-based host cell reactivation assays, persistence of ionizing radiation-induced DNA double-strand breaks and cellular hypersensitivity to ionizing radiation. Mechanistically, we find that nickel, in contrast to the metalloid arsenic, acutely induces transcriptional repression of HDR and MMR genes as part of a global transcriptional pattern similar to that seen with hypoxia. Finally, we find that exposure to low-dose nickel reduces the activity of the MLH1 promoter, but only arsenic leads to long-term MLH1 promoter silencing. Together, our data elucidate novel mechanisms of heavy metal carcinogenesis and contribute to our understanding of the influence of the microenvironment on the regulation of DNA repair pathways. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Role of XRCC4 phosphorylation by DNA-PK in the regulation of NHEJ repair pathway of DNA double strand break

    International Nuclear Information System (INIS)

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Kamdar, Radhika P.; Sicheng, Liu; Wanotayan, Rujira; Matsumoto, Yoshihisa

    2014-01-01

    Non-homologous end-joining (NHEJ) is the predominant pathway of DNA double strand breaks in higher eukaryotes and is active throughout the cell cycle. NHEJ repair includes many factors as Ku70/86, DNA-PKcs, XRCC4-Ligase IV complex and XLF (also known as Cernunnos). In these factors, DNA-PKcs acts as central regulator in NHEJ repair. It recruited at the DNA damages site after DNA damage and after association with Ku its kinase activity is activated. It phosphorylates many of important NHEJ proteins in vitro including XRCC4, Ku 70/86, Artemis, and even DNA-PKcs but till now, very less studies have been done to know the role and significance of phosphorylation in the NHEJ repair. Studies by other researchers identified various phosphorylation sites in XRCC4 by DNA-PK using mass spectrometry but these phosphorylation sites were shown to be dispensable for DSB repair. In the present investigation, we identified 3 serine and one new threonine phosphorylation sites in XRCC4 protein by DNA-PK. In vivo phosphorylation at these sites was verified by generating phosphorylation specific antibodies and the requirement for DNA-PK therein was verified by using DNA-PK inhibitor and DNA-PK proficient and deficient cell lines in response to radiation and zeocin treatment. We have also found that phosphorylation at these sites showed dose dependency in response to radiation treatment. The two serine and one threonine phosphorylation site is also biological important as their mutation into alanine significantly elevated radiosensitivity as measured by colony formation assay. Neutral comet assay showed delayed kinetics in DSB repair of these mutants. Furthermore, we have found a protein, with putative DSB repair function, which interacts with domain including the phosphorylation sites.These results indicate that these phosphorylation sites would mediate functional link between XRCC4 and DNA-PK. (author)

  8. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    International Nuclear Information System (INIS)

    Kim, Hyun-Suk; Guo, Chunlu; Thompson, Eric L.; Jiang, Yanlin; Kelley, Mark R.; Vasko, Michael R.; Lee, Suk-Hee

    2015-01-01

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons

  9. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Suk [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Guo, Chunlu; Thompson, Eric L. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Jiang, Yanlin [Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Lee, Suk-Hee, E-mail: slee@iu.edu [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States)

    2015-09-15

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

  10. International congress on DNA damage and repair: Book of abstracts

    Energy Technology Data Exchange (ETDEWEB)

    1987-01-01

    This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

  11. International congress on DNA damage and repair: Book of abstracts

    International Nuclear Information System (INIS)

    1987-01-01

    This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation

  12. DNA Repair Systems

    Indian Academy of Sciences (India)

    Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

  13. Emerging roles of the nucleolus in regulating the DNA damage response: the noncanonical DNA repair enzyme APE1/Ref-1 as a paradigmatical example.

    Science.gov (United States)

    Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia; Tell, Gianluca

    2014-02-01

    An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world.

  14. DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity

    DEFF Research Database (Denmark)

    Gupta, Rajat; Somyajit, Kumar; Narita, Takeo

    2018-01-01

    Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expres...... and the evolution of antibody CSR in higher vertebrates....

  15. 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

  16. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during...... recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits...

  17. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

    International Nuclear Information System (INIS)

    Sebastian, J.; Sancar, G.B.

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

  18. FANCD2 Binds CtIP and Regulates DNA-End Resection during DNA Interstrand Crosslink Repair

    Directory of Open Access Journals (Sweden)

    Junya Unno

    2014-05-01

    Full Text Available The Fanconi anemia (FA pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs, where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMC-induced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair.

  19. Radiobiological significance of DNA repair

    International Nuclear Information System (INIS)

    Kuzin, A.M.

    1978-01-01

    A short outline is given on the history of the problem relating to the repair of radiation injuries, specifically its molecular mechanisms. The most urgent problems which currently confront the researchers are noted. This is a further study on the role of DNA repair in post-radiation recovery, search for ways to activate and suppress DNA repair, investigations into the activity balance of various repair enzymes as well as the problem of errors in the structure of repairing DNA. An important role is attached to the investigations of DNA repair in solving a number of practical problems

  20. Beyond DNA repair: DNA-PK function in cancer

    OpenAIRE

    Goodwin, Jonathan F.; Knudsen, Karen E.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, furthe...

  1. The journey of DNA repair.

    Science.gov (United States)

    Saini, Natalie

    2015-12-01

    21 years ago, the DNA Repair Enzyme was declared "Molecule of the Year". Today, we are celebrating another "year of repair", with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  2. My journey to DNA repair.

    Science.gov (United States)

    Lindahl, Tomas

    2013-02-01

    I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mammalian exonucleases DNase III (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O(6)-methylguanine (O(6)mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation. Copyright © 2013. Production and hosting by Elsevier Ltd.

  3. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...... base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby...

  4. NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

    Science.gov (United States)

    Volcic, Meta; Karl, Sabine; Baumann, Bernd; Salles, Daniela; Daniel, Peter; Fulda, Simone; Wiesmüller, Lisa

    2012-01-01

    NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

  5. The journey of DNA repair

    OpenAIRE

    Saini, Natalie

    2015-01-01

    21 years ago, the DNA Repair Enzyme was declared “Molecule of the Year”. Today, we are celebrating another “year of repair”, with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  6. Fanconi anemia and DNA repair.

    Science.gov (United States)

    Grompe, M; D'Andrea, A

    2001-10-01

    Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects. The principal cellular phenotype is hypersensitivity to DNA damage, particularly interstrand DNA crosslinks. The FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. Five of the FA proteins (FANCA, C, E, F and G) interact in a nuclear complex upstream of FANCD2. FANCB and FANCD1 have not yet been cloned, but it is likely that FANCB is part of the nuclear complex and that FANCD1 acts downstream of FANCD2. The FA nuclear complex regulates the mono-ubiquitination of FANCD2 in response to DNA damage, resulting in targeting of this protein into nuclear foci. These foci also contain BRCA1 and other DNA damage response proteins. In male meiosis, FANCD2 also co-localizes with BRCA1 at synaptonemal complexes. Together, these data suggest that the FA pathway functions primarily as a DNA damage response system, although its exact role (direct involvement in DNA repair versus indirect, facilitating role) has not yet been defined.

  7. Monogenic diseases of DNA repair

    DEFF Research Database (Denmark)

    Keijzers, Guido; Bakula, Daniela; Scheibye-Knudsen, Morten

    2017-01-01

    Maintaining the stability of the genome is essential for all organisms, and it is not surprising that damage to DNA has been proposed as an explanation for multiple chronic diseases.1-5 Conserving a pristine genome is therefore of central importance to our health. To overcome the genotoxic stress...... of a growing number of human diseases. Notably, many of these monogenic DNA-repair disorders display features of accelerated aging, supporting the notion that genome maintenance is a key factor for organismal longevity. This review focuses on the physiological consequences of loss of DNA repair, particularly...... in the context of monogenic DNA-repair diseases....

  8. Structural aspects of DNA in its replication and repair

    International Nuclear Information System (INIS)

    Mitra, S.; Pal, B.C.; Foote, R.S.; Bates, R.C.; Bhattacharyya, A.; Snow, E.T.; Wobbe, C.R.; Morse, C.C.; Snyder, C.E.

    1984-01-01

    The research objective of this laboratory is to investigate the structure of DNA, the mechanism of DNA replication and its regulation, and the mechanism and role of repair of the altered DNA in the expression of heritable changes. This research has two broad aims, namely investigation of (a) the regulation of DNA replication in mammals, using parvovirus DNA as a model system and (b) the role of DNA repair in mutagenesis and carcinogenesis induced by simple alkylating mutagens

  9. Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.; Bodell, W.J.; Morgan, W.F.; Zelle, B.

    1983-08-10

    Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair. Previous claims that 3-aminobenzamide stimulates repair synthesis after exposure to UV light are probably artifacts, because the stimulations are only observed in lymphocytes in the presence of a high concentration of hydroxyurea that itself inhibits repair. The initial inhibition of semiconservative DNA synthesis and the excision of the major alkylation products and pyrimidine dimers were unaffected by 3-aminobenzamide. In general poly(ADP-ribose) synthesis appears to be uniquely involved in regulating the ligation stage of repair of alkylation damage but not ultraviolet damage. By regulating the ligation efficiency, poly(ADP-ribosylation) modulates the dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks. The ligation stage of repair of UV damage appears different and is not regulated by poly(ADP-ribosylation).

  10. Role of Cell Cycle Regulation and MLH1, A Key DNA Mismatch Repair Protein, In Adaptive Survival Responses. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    David A. Boothman

    1999-08-11

    Due to several interesting findings on both adaptive survival responses (ASRs) and DNA mismatch repair (MMR), this grant was separated into two discrete Specific Aim sets (each with their own discrete hypotheses). The described experiments were simultaneously performed.

  11. Aging and DNA repair capability. [Review

    Energy Technology Data Exchange (ETDEWEB)

    Tice, R R

    1977-01-01

    A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

  12. Mitochondrial DNA repair and aging

    International Nuclear Information System (INIS)

    Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

    2002-01-01

    The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis

  13. Mitochondrial DNA repair and aging

    Energy Technology Data Exchange (ETDEWEB)

    Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

    2002-11-30

    The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis.

  14. DNA repair in human cells

    International Nuclear Information System (INIS)

    Regan, J.D.; Carrier, W.L.; Kusano, I.; Furuno-Fukushi, I.; Dunn, W.C. Jr.; Francis, A.A.; Lee, W.H.

    1982-01-01

    Our primary objective is to elucidate the molecular events in human cells when cellular macromolecules such as DNA are damaged by radiation or chemical agents. We study and characterize (i) the sequence of DNA repair events, (ii) the various modalities of repair, (iii) the genetic inhibition of repair due to mutation, (iv) the physiological inhibition of repair due to mutation, (v) the physiological inhibition of repair due to biochemical inhibitors, and (vi) the genetic basis of repair. Our ultimate goals are to (i) isolate and analyze the repair component of the mutagenic and/or carcinogenic event in human cells, and (ii) elucidate the magnitude and significance of this repair component as it impinges on the practical problems of human irradiation or exposure to actual or potential chemical mutagens and carcinogens. The significance of these studies lies in (i) the ubiquitousness of repair (most organisms, including man, have several complex repair systems), (ii) the belief that mutagenic and carcinogenic events may arise only from residual (nonrepaired) lesions or that error-prone repair systems may be the major induction mechanisms of the mutagenic or carcinogenic event, and (iii) the clear association of repair defects and highly carcinogenic disease states in man [xeroderma pigmentosum (XP)

  15. Mammalian DNA Repair. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Wood, Richard D.

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  16. CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

    International Nuclear Information System (INIS)

    Desai, Amar; Webb, Bryan; Gerson, Stanton L.

    2014-01-01

    Background: Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells. Materials and methods: A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ϒ-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes. Results: A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells. Conclusions: CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs

  17. Down-regulation of DNA mismatch repair enhances initiation and growth of neuroblastoma and brain tumour multicellular spheroids.

    Directory of Open Access Journals (Sweden)

    Samuel L Collins

    Full Text Available Multicellular tumour spheroid (MCTS cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR. This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.

  18. Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic Escherichia coli.

    Science.gov (United States)

    Molina-Quiroz, Roberto C; Silva-Valenzuela, Cecilia; Brewster, Jennifer; Castro-Nallar, Eduardo; Levy, Stuart B; Camilli, Andrew

    2018-01-09

    Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli , as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli IMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli , the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds. Copyright © 2018 Molina-Quiroz et al.

  19. Smurf2 Regulates DNA Repair and Packaging to Prevent Tumors | Center for Cancer Research

    Science.gov (United States)

    The blueprint for all of a cell’s functions is written in the genetic code of DNA sequences as well as in the landscape of DNA and histone modifications. DNA is wrapped around histones to package it into chromatin, which is stored in the nucleus. It is important to maintain the integrity of the chromatin structure to ensure that the cell continues to behave appropriately.

  20. Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

    Science.gov (United States)

    Pennisi, Rosa; Antoccia, Antonio; Leone, Stefano; Ascenzi, Paolo; di Masi, Alessandra

    2017-08-01

    The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair. © 2017 Federation of European Biochemical Societies.

  1. Molecular biological mechanisms I. DNA repair

    International Nuclear Information System (INIS)

    Friedl, A.A.

    2000-01-01

    Cells of all living systems possess a variety of mechanisms that allow to repair spontaneous and exogeneously induced DNA damage. DNA repair deficiencies may invoke enhanced sensitivity towards DNA-damaging agents such as ionizing radiation. They may also enhance the risk of cancer development, both spontaneously or after induction. This article reviews several DNA repair mechanisms, especially those dealing with DNA double-strand breaks, and describes hereditary diseases associated with DNA repair defects. (orig.) [de

  2. A lncRNA to repair DNA

    DEFF Research Database (Denmark)

    Lukas, Jiri; Altmeyer, Matthias

    2015-01-01

    Long non-coding RNAs (lncRNAs) have emerged as regulators of various biological processes, but to which extent lncRNAs play a role in genome integrity maintenance is not well understood. In this issue of EMBO Reports, Sharma et al [1] identify the DNA damage-induced lncRNA DDSR1 as an integral...... player of the DNA damage response (DDR). DDSR1 has both an early role by modulating repair pathway choices, and a later function when it regulates gene expression. Sharma et al [1] thus uncover a dual role for a hitherto uncharacterized lncRNA during the cellular response to DNA damage....

  3. Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells1

    Science.gov (United States)

    Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

    2007-01-01

    Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse LβT2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of LβT2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature αT3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA. PMID:15128600

  4. Gonadotropin-releasing hormone regulates expression of the DNA damage repair gene, Fanconi anemia A, in pituitary gonadotroph cells.

    Science.gov (United States)

    Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

    2004-09-01

    Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse L beta T2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of L beta T2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature alpha T3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

  5. Decreased cell survival and DNA repair capacity after UVC irradiation in association with down-regulation of GRP78/BiP in human RSa cells

    International Nuclear Information System (INIS)

    Zhai Ling; Kita, Kazuko; Wano, Chieko; Wu Yuping; Sugaya, Shigeru; Suzuki, Nobuo

    2005-01-01

    In contrast to extensive studies on the roles of molecular chaperones, such as heat shock proteins, there are only a few reports about the roles of GRP78/BiP, an endoplasmic reticulum (ER) stress-induced molecular chaperone, in mammalian cell responses to DNA-damaging stresses. To investigate whether GRP78/BiP is involved in resistance to a DNA-damaging agent, UVC (principally 254 nm in wavelength), we established human cells with down-regulation of GRP78/BiP by transfection of human RSa cells with antisense cDNA for GRP78/BiP. We found that the transfected cells showed higher sensitivity to UVC-induced cell death than control cells transfected with the vector alone. In the antisense-cDNA transfected cells, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4) photoproducts) in vivo and DNA synthesis activity of whole cell extracts to repair UVC-irradiated plasmids in vitro were remarkably decreased compared with those in the control cells. Furthermore, the antisense-cDNA transfected cells also showed slightly higher sensitivity to cisplatin-induced cell death than the control cells. Cisplatin-induced DNA damage is primarily repaired by nucleotide excision repair, like UVC-induced DNA damage. The present results suggest that GRP78/BiP plays a protective role against UVC-induced cell death possibly via nucleotide excision repair, at least in the human RSa cells tested

  6. DNA repair in PHA stimulated human lymphocytes

    International Nuclear Information System (INIS)

    Catena, C.; Mattoni, A.

    1984-01-01

    Damage an repair of radiation induced DNA strand breaks were measured by alkaline lysis and hydroxyapatite chromatography. PHA stimulated human lymphocytes show that the rejoining process is complete within the first 50 min., afterwords secondary DNA damage and chromatid aberration. DNA repair, in synchronized culture, allows to evaluate individual repair capacity and this in turn can contribute to the discovery of individual who, although they do not demonstrate apparent clinical signs, are carriers of DNA repair deficiency. Being evident that a correlation exists between DNA repair capacity and carcinogenesis, the possibility of evaluating the existent relationship between DNA repair and survival in tumor cells comes therefore into discussion

  7. DNA Repair, Redox Regulation and Modulation of Estrogen Receptor Alpha Mediated Transcription

    Science.gov (United States)

    Curtis-Ducey, Carol Dianne

    2009-01-01

    Interaction of estrogen receptor [alpha] (ER[alpha]) with 17[beta]-estradiol (E[subscript 2]) facilitates binding of the receptor to estrogen response elements (EREs) in target genes, which in turn leads to recruitment of coregulatory proteins. To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of…

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-05

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

  10. Mutagenic DNA repair in enterobacteria

    International Nuclear Information System (INIS)

    Sedgwick, S.G.; Chao Ho; Woodgate, R.

    1991-01-01

    Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD' fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD' antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium u mu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umuDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention

  11. Cloning human DNA repair genes

    International Nuclear Information System (INIS)

    Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

    1994-01-01

    Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

  12. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

  13. 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.

  14. DNA Repair and Genome Maintenance in Bacillus subtilis

    Science.gov (United States)

    Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

  15. Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

    Science.gov (United States)

    Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H; Rao, Jasti S

    2011-01-01

    Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand

  16. Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

    Directory of Open Access Journals (Sweden)

    Shivani Ponnala

    Full Text Available Glioblastoma Multiforme (GBM is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ repair mechanism plays a major role in double strand break (DSB repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU and MMP9-cathepsin B (pMC shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from

  17. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    International Nuclear Information System (INIS)

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-01-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  18. GANP regulates the choice of DNA repair pathway by DNA-PKcs interaction in AID-dependent IgV region diversification.

    Science.gov (United States)

    Eid, Mohammed Mansour Abbas; Maeda, Kazuhiko; Almofty, Sarah Ameen; Singh, Shailendra Kumar; Shimoda, Mayuko; Sakaguchi, Nobuo

    2014-06-15

    RNA export factor germinal center-associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining-proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment. Copyright © 2014 by The American Association of Immunologists, Inc.

  19. Recent advances in DNA repair and recombination.

    Science.gov (United States)

    Iwanejko, L A; Jones, N J

    1998-09-11

    The subjects of the talks at this 1-day DNA Repair Network meeting, held at City University, London on December 15, 1997, encompassed a range of topics and reflected some of the current areas of research in the United Kingdom. Topics included DNA double-strand break repair, V(D)J recombination, DNA ligases, the RecQ family of helicases and Bloom's syndrome, UVB and immunosuppression, the repair of oxidative damage and mismatch repair mechanisms.

  20. p53 regulates the repair of DNA double-strand breaks by both homologous and non-homologous recombination

    International Nuclear Information System (INIS)

    Willers, H.; Powell, S.N.; Dahm-Daphi, J.

    2003-01-01

    Full text: p53 is known to suppress spontaneous homologous recombination (HR), while its role in non-homologous recombination (NHR) remains to be clarified. Here, we sought to determine the influence of p53 on the repair of chromosomal double-strand breaks (DSBs) by HR or NHR using specially designed recombination substrates that integrate into the genome. Isogenic mouse fibroblast pairs with or without expression of exogenous p53 protein were utilized. A reporter plasmid carrying a mutated XGPRT gene was chromosomally integrated and DSBs were generated within the plasmid by the I-SceI endonuclease. Subsequent homology-mediated repair from an episomal donor resulted in XGPRT reconstitution and cellular resistance to a selection antibiotic. Analogously, the repair of chromosomal I-SceI breaks by NHR using another novel reporter plasmid restored XGPRT translation. For p53-null cells, the mean frequency of I-SceI break repair via HR was 5.5 x 10 -4 . The p53-Val135 mutant, which previously has been shown to suppress spontaneous HR by 14-fold employing the same cell system and reporter gene, only caused a 2- to 3-fold suppression of break-induced HR. In contrast, a dramatic effect of p53 on repair via NHR was found. Preliminary sequence analysis indicated that there was at least a 1000-fold reduction of illegitimate repair events resulting in loss of sequence at the break sites. The observed effects were mediated by p53 mutants defective in regulation of the cell-cycle and apoptosis. The main findings were: (1) p53 virtually blocked illegitimate rejoining of chromosomal ends. (2) The suppression of homologous DSB repair was less pronounced than the inhibition of spontaneous HR. We hypothesize that p53 allows to a certain extent error-free homology-dependent repair to proceed, while blocking error-prone NHR. The data support and extent a previous model, in which p53 maintains genomic stability by regulating recombination independently of its transactivation function

  1. 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

  2. Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents.

    Science.gov (United States)

    Francia, Giulio; Green, Shane K; Bocci, Guido; Man, Shan; Emmenegger, Urban; Ebos, John M L; Weinerman, Adina; Shaked, Yuval; Kerbel, Robert S

    2005-10-01

    Similar to other anticancer agents, intrinsic or acquired resistance to DNA-damaging chemotherapeutics is a major obstacle for cancer therapy. Current strategies aimed at overcoming this problem are mostly based on the premise that tumor cells acquire heritable genetic mutations that contribute to drug resistance. Here, we present evidence for an epigenetic, tumor cell adhesion-mediated, and reversible form of drug resistance that is associated with a reduction of DNA mismatch repair proteins PMS2 and/or MLH1 as well as other members of this DNA repair process. Growth of human breast cancer, human melanoma, and murine EMT-6 breast cancer cell lines as multicellular spheroids in vitro, which is associated with increased resistance to many chemotherapeutic drugs, including alkylating agents, is shown to lead to a reproducible down-regulation of PMS2, MLH1, or, in some cases, both as well as MHS6, MSH3, and MSH2. The observed down-regulation is in part reversible by treatment of tumor spheroids with the DNA-demethylating agent, 5-azacytidine. Thus, treatment of EMT-6 mouse mammary carcinoma spheroids with 5-azacytidine resulted in reduced and/or disrupted cell-cell adhesion, which in turn sensitized tumor spheroids to cisplatin-mediated killing in vitro. Our results suggest that antiadhesive agents might sensitize tumor spheroids to alkylating agents in part by reversing or preventing reduced DNA mismatch repair activity and that the chemosensitization properties of 5-azacytidine may conceivably reflect its role as a potential antiadhesive agent as well as reversal agent for MLH1 gene silencing in human tumors.

  3. Endogenous DNA Damage and Repair Enzymes

    Directory of Open Access Journals (Sweden)

    Arne Klungland

    2016-06-01

    Full Text Available Tomas Lindahl completed his medical studies at Karolinska Institute in 1970. Yet, his work has always been dedicated to unraveling fundamental mechanisms of DNA decay and DNA repair. His research is characterized with groundbreaking discoveries on the instability of our genome, the identification of novel DNA repair activities, the characterization of DNA repair pathways, and the association to diseases, throughout his 40 years of scientific career.

  4. Pim-3 contributes to radioresistance through regulation of the cell cycle and DNA damage repair in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiang-Yuan; Wang, Zhen [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Bei [Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Ying-Jian, E-mail: yjzhang111@aliyun.com [Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Ying-Yi, E-mail: liyingyi@fudan.edu.cn [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China)

    2016-04-22

    Resistance of cancer cells to chemoradiotherapy is a major clinical problem in pancreatic cancer treatment. Therefore, understanding the molecular basis of cellular resistance and identifying novel targets are essential for improving treatment efficacy for pancreatic cancer patients. Previous studies have demonstrated a significant role for Pim-3 in pancreatic cancer survival against gemcitabine-induced genotoxic stress. Here, we observed that radiation treatment enhanced Pim-3 expression in human pancreatic cancer cells in vitro. Stable overexpression of Pim-3 in pancreatic cancer cells significantly protected cells against radiation treatment by attenuating G2/M phase cell cycle arrest and DNA damage response. Silencing of Pim-3 expression significantly elevated the phosphorylation of histone variant H2AX, a marker of DNA double strand breaks, and decreased the activation of ataxia-telangiectasia-mutated (ATM) kinase, along with its downstream targets, eventually enhancing the radiosensitivity of human pancreatic cancer cells in vitro and in vivo. Hence, we demonstrated a novel function for Pim-3 in human pancreatic cancer cell survival against radiation. Targeting Pim-3 may be a promising way to improve treatment efficacy in combination with radiotherapy in human pancreatic cancer. - Highlights: • This is first study to demonstrate that Pim-3 is endogenously induced by ionizing radiation in pancreatic cancer cells, and Pim-3 overexpression enhanced radioresistance of pancreatic cancer cells both in vitro and in vivo. • This is first study to provide evidence that radioresistance induced by Pim-3 is mainly attributed to Pim-3 induces activation of ATM, which subsequently activates checkpoint 1, leading to amplification of DNA repair through cell cycle arrest and DNA repair pathways. • This is first study to indicate that targeting Pim-3 may be a promising strategy to provide better treatment efficacy in combination with radiotherapy in human pancreatic

  5. Pim-3 contributes to radioresistance through regulation of the cell cycle and DNA damage repair in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Chen, Xiang-Yuan; Wang, Zhen; Li, Bei; Zhang, Ying-Jian; Li, Ying-Yi

    2016-01-01

    Resistance of cancer cells to chemoradiotherapy is a major clinical problem in pancreatic cancer treatment. Therefore, understanding the molecular basis of cellular resistance and identifying novel targets are essential for improving treatment efficacy for pancreatic cancer patients. Previous studies have demonstrated a significant role for Pim-3 in pancreatic cancer survival against gemcitabine-induced genotoxic stress. Here, we observed that radiation treatment enhanced Pim-3 expression in human pancreatic cancer cells in vitro. Stable overexpression of Pim-3 in pancreatic cancer cells significantly protected cells against radiation treatment by attenuating G2/M phase cell cycle arrest and DNA damage response. Silencing of Pim-3 expression significantly elevated the phosphorylation of histone variant H2AX, a marker of DNA double strand breaks, and decreased the activation of ataxia-telangiectasia-mutated (ATM) kinase, along with its downstream targets, eventually enhancing the radiosensitivity of human pancreatic cancer cells in vitro and in vivo. Hence, we demonstrated a novel function for Pim-3 in human pancreatic cancer cell survival against radiation. Targeting Pim-3 may be a promising way to improve treatment efficacy in combination with radiotherapy in human pancreatic cancer. - Highlights: • This is first study to demonstrate that Pim-3 is endogenously induced by ionizing radiation in pancreatic cancer cells, and Pim-3 overexpression enhanced radioresistance of pancreatic cancer cells both in vitro and in vivo. • This is first study to provide evidence that radioresistance induced by Pim-3 is mainly attributed to Pim-3 induces activation of ATM, which subsequently activates checkpoint 1, leading to amplification of DNA repair through cell cycle arrest and DNA repair pathways. • This is first study to indicate that targeting Pim-3 may be a promising strategy to provide better treatment efficacy in combination with radiotherapy in human pancreatic

  6. Mutagenesis and repair of DNA

    International Nuclear Information System (INIS)

    Janion, C.; Grzesiuk, E.; Fabisiewicz, A.; Tudek, B.; Ciesla, J.; Graziewicz, M.; Wojcik, A.; Speina, E.

    1998-01-01

    Full text. The discovery that the mfd gene codes for a transcription-coupling repair factor (TRCF) prompted us to re-investigate the MFD (mutation frequency decline) phenomenon in E.coli K-12 strain when mutations were induced by ultraviolet light, halogen light or MMS-treatment. These studies revealed that: (i) the process of MFD involves the proofreading activity of DNA pol III and the mismatch repair system, as well as, TRCF and the UvrABC-excinuclease (ii) a semi-rich plate test may be replaced by a rich liquid medium, (iii) the T-T pyrimidine dimers are the lesions excised with the highest activity, and (iv) overproduction of UmuD(D'C) proteins leads to a great increase in mutant frequency in irradiated and MMS-treated cells. The role of mismatch repair (MR) in MMS-induced mutagenesis is obscured by the fact that the spectra of mutational specificity are different in bacteria proficient and deficient in MR. It has been found that transposons Tn10 (and Tn5) when inserted into chromosomal DNA of E. coli influence the phenotype lowering the survival and frequency of mutations induced by UV or halogen light irradiation. This is connected with a deficiency of UmuD(D') and UmuC proteins. Transformation of bacteria with plasmids bearing the umuD(D')C genes, suppresses the effects of the transposon insertion, a phenomenon which has not been described before. Single-stranded DNA of M13mp18 phage was oxidized in vitro by a hydroxyl radical generating system including hypoxanthine/xanthine oxidase/Fe3+/EDTA, and it was found that Fapy-Ade, Fapy-Gua, 8-oxyAde and thymine glycol were the main products formed. Replication of the oxidized template by T7 phage DNA polymerase, Klenow fragment of polymerase I, or polymerase beta from bovine thymus has revealed that oxidized pyrimidines are stronger blockers than oxidized purines for T7 phage and Klenow fragment polymerases and the blocking potency depends on the neighboring bases and on the type of polymerase. Studies of

  7. Oxidative DNA damage & repair: An introduction.

    Science.gov (United States)

    Cadet, Jean; Davies, Kelvin J A

    2017-06-01

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

  8. DNA damage and repair in plants

    International Nuclear Information System (INIS)

    Britt, A.B.

    1996-01-01

    The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

  9. DNA repair in non-mammalian animals

    International Nuclear Information System (INIS)

    Mitani, Hiroshi

    1984-01-01

    Studies on DNA repair have been performed using microorganisms such as Escherichia coli and cultured human and mammalian cells. However, it is well known that cultured organic cells differ from each other in many respects, although DNA repair is an extremely fundamental function of organisms to protect genetic information from environmental mutagens such as radiation and 0 radicals developing in the living body. To answer the question of how DNA repair is different between the animal species, current studies on DNA repair of cultured vertebrate cells using the methods similar to those in mammalian experiments are reviewed. (Namekawa, K.)

  10. DNA repair in Mycobacterium tuberculosis revisited.

    Science.gov (United States)

    Dos Vultos, Tiago; Mestre, Olga; Tonjum, Tone; Gicquel, Brigitte

    2009-05-01

    Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr - proteins previously presumed to be produced exclusively in mammalian cells - and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.

  11. 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)

  12. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  13. DNA damage repair and radiosensitivity

    International Nuclear Information System (INIS)

    Suzuki, Norio

    2003-01-01

    Tailored treatment is not new in radiotherapy; it has been the major subject for the last 20-30 years. Radiation responses and RBE (relative biological effectiveness) depend on assay systems, endpoints, type of tissues and tumors, radiation quality, dose rate, dose fractionation, physiological and environmental factors etc, Latent times to develop damages also differ among tissues and endpoints depending on doses and radiation quality. Recent progress in clarification of radiation induced cell death, especially of apoptotic cell death, is quite important for understanding radiosensitivity of tumor cure process as well as of tumorigenesis. Apoptotic cell death as well as dormant cells had been unaccounted and missed into a part of reproductive cell death. Another area of major progress has been made in clarifying repair mechanisms of radiation damage, i.e., non-homologous end joining (NHEJ) and homologous recombinational repair (HRR). New approaches and developments such as cDNA or protein micro arrays and so called informatics in addition to basic molecular biological analysis are expected to aid identifying molecules and their roles in signal transduction pathways, which are multi-factorial and interactive each other being involved in radiation responses. (authors)

  14. Metabolic modulation of mammalian DNA excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Schrader, T.J.

    1988-01-01

    First, ultraviolet light (UVL)- and dimethylsulfate (DMS)-induced excision repair was examined in quiescent and lectin-stimulated bovine lymphocytes. Upon mitogenic stimulation, UVL-induced repair increased by a factor of 2 to 3, and reached this maximum 2 days before the onset of DNA replication. However, DMS-induced repair increased sevenfold in parallel with DNA replication. Repair patch sizes were smaller for DMS-induced damage reflecting patches of 7 nucleotides in quiescent lymphocytes compared to 20 nucleotides induced by UVL. The patch size increased during lymphocyte stimulation until one day prior to the peak of DNA replication when patch sizes of 45 and 35 nucleotides were produced in response to UVL- and DMS-induced damage, respectively. At the peak of DNA replication, the patch sizes were equal for both damaging agents at 34 nucleotides. In the second study, a small amount of repair replication was observed in undamaged quiescent and concanavalin A-stimulated bovine lymphocytes as well as in human T98G glioblastoma cells. Repair incorporation doubled in the presence of hydroxyurea. Thirdly, the enhanced repair replication induced by the poly (ADP-ribose) polymerase inhibitor, 3-aminobenzamide, (3-AB), could not be correlated either with an increased rate of repair in the presence of 3-AB or with the use of hydroxyurea in the repair protocol. Finally, treatment of unstimulated lymphocytes with hyperthermia was accompanied by decreased repair replication while the repair patches remained constant at 20 nucleotides.

  15. DNA repair: Dynamic defenders against cancer and aging

    Energy Technology Data Exchange (ETDEWEB)

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

    You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet

  16. DNA repair phenotype and dietary antioxidant supplementation

    DEFF Research Database (Denmark)

    Guarnieri, Serena; Loft, Steffen; Riso, Patrizia

    2008-01-01

    Phytochemicals may protect cellular DNA by direct antioxidant effect or modulation of the DNA repair activity. We investigated the repair activity towards oxidised DNA in human mononuclear blood cells (MNBC) in two placebo-controlled antioxidant intervention studies as follows: (1) well-nourished......Phytochemicals may protect cellular DNA by direct antioxidant effect or modulation of the DNA repair activity. We investigated the repair activity towards oxidised DNA in human mononuclear blood cells (MNBC) in two placebo-controlled antioxidant intervention studies as follows: (1) well......-nourished subjects who ingested 600 g fruits and vegetables, or tablets containing the equivalent amount of vitamins and minerals, for 24 d; (2) poorly nourished male smokers who ingested 500 mg vitamin C/d as slow- or plain-release formulations together with 182 mg vitamin E/d for 4 weeks. The mean baseline levels...

  17. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Aman Wang

    2017-05-01

    Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

  18. Repair of DNA in xeroderma pigmentosum conjunctiva

    International Nuclear Information System (INIS)

    Newsome, D.A.; Kraemer, K.H.; Robbins, J.H.

    1975-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive disease with tumor formation on sun-exposed areas of the skin and eyes. Cells from most XP patients are deficient in repairing DNA damaged by ultraviolet (uv) light as shown by a reduced rate of tritiated thymidine (3HTdR) incorporation during their DNA repair synthesis. We have studied such repair synthesis in conjunctival cells from an XP patient with a conjunctival epithelioma and from normal cadaver conjunctiva. Cultured conjunctival cells were irradiated with uv light and then incubated with 3HTdR. Autoradiograms were prepared and showed that uv radiation induced a considerably slower rate of DNA repair synthesis in the XP cells than in normal cells. Many of the ocular abnormalities of XP, including tumor formation, may be the result of this defective DNA repair process

  19. Role of Cell Cycle Regulation and MLH1, A Key DNA Mismatch Repair Protein, In Adaptive Survival Responses. Final Report; FINAL

    International Nuclear Information System (INIS)

    David A. Boothman

    1999-01-01

    Due to several interesting findings on both adaptive survival responses (ASRs) and DNA mismatch repair (MMR), this grant was separated into two discrete Specific Aim sets (each with their own discrete hypotheses). The described experiments were simultaneously performed

  20. DNA N-glycosylases and uv repair

    Energy Technology Data Exchange (ETDEWEB)

    Demple, B; Linn, S

    1980-09-18

    Repair of some DNA photoproducts can be mediated by glycosylic bond hydrolysis. Thus, Escherichia coli endonuclease III releases 5,6-hydrated thymines as free bases, while T4 uv endonuclease releases one of two glycosylic bonds holding pyrimidine dimers in DNA. In contrast, uninfected E. coli apparently does not excise pyrimidine dimers via a DNA glycosylase.

  1. Repair of abasic sites in DNA

    Energy Technology Data Exchange (ETDEWEB)

    Dianov, Grigory L.; Sleeth, Kate M.; Dianova, Irina I.; Allinson, Sarah L

    2003-10-29

    Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase {beta} adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase {delta}/{epsilon} and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase {delta}/{epsilon} is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.

  2. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao

    2011-01-01

    Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms.While bacteria require only three proteins to complete the incision step of NER,eukaryotes employ about 30 proteins to complete the same step.Here we summarize recent studies demonstrating that ubiquitination,a post-translational modification,plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis.Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process.We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

  3. Human diseases associated with defective DNA repair

    International Nuclear Information System (INIS)

    Friedberg, E.C.; Ehmann, U.K.; Williams, J.I.

    1979-01-01

    The observations on xeroderma pigmentosum (XP) cells in culture were the first indications of defective DNA repair in association with human disease. Since then, a wealth of information on DNA repair in XP, and to a lesser extent in other diseases, has accumulated in the literature. Rather than clarifying the understanding of DNA repair mechanisms in normal cells and of defective DNA repair in human disease, the literature suggests an extraordinary complexity of both of the phenomena. In this review a number of discrete human diseases are considered separately. An attempt was made to systematically describe the pertinent clinical features and cellular and biochemical defects in these diseases, with an emphasis on defects in DNA metabolism, particularly DNA repair. Wherever possible observations have been correlated and unifying hypotheses presented concerning the nature of the basic defect(s) in these diseases. Discussions of the following diseases are presented: XP, ataxia telangiectasia; Fanconi's anemia; Hutchinson-Gilford progeria syndrome; Bloom's syndrome, Cockayne's syndrome; Down's syndrome; retinoblastoma; chronic lymphocytic leukemia; and other miscellaneous human diseases with possble DNA repair defects

  4. Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

    Science.gov (United States)

    Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

    2014-01-01

    Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

  5. Hsp90: A New Player in DNA Repair?

    Directory of Open Access Journals (Sweden)

    Rosa Pennisi

    2015-10-01

    Full Text Available Heat shock protein 90 (Hsp90 is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis, transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR pathways that include: (i cell cycle arrest; (ii transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.

  6. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.

    1988-01-01

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  7. DNA repair related to radiation therapy

    International Nuclear Information System (INIS)

    Klein, W.

    1979-01-01

    The DNA excision repair capacity of peripheral human lymphocytes after radiation therapy has been analyzed. Different forms of application of the radiation during the therapy have been taken into account. No inhibition of repair was found if cells were allowed a certain amount of accomodation to radiation, either by using lower doses or longer application times. (G.G.)

  8. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  9. Small molecules, inhibitors of DNA-PK, targeting DNA repair and beyond

    Directory of Open Access Journals (Sweden)

    David eDavidson

    2013-01-01

    Full Text Available Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining (NHEJ a major mechanism for the repair of double strand breaks (DSB in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK. The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA

  10. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  11. Recombinational DNA repair and human disease

    International Nuclear Information System (INIS)

    Thompson, Larry H.; Schild, David

    2002-01-01

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities

  12. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    National Research Council Canada - National Science Library

    Goldman, Radoslav

    2008-01-01

    .... To evaluate this hypothesis we quantify DNA repair capacity in blood cells using comet assay and evaluate how this repair capacity is related to genetic variants in OGG1 and XRCC1 DNA repair genes...

  13. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    National Research Council Canada - National Science Library

    Goldman, Radoslav

    2007-01-01

    .... To evaluate this hypothesis we quantify DNA repair capacity in blood cells using comet assay and evaluate how this repair capacity is related to genetic variants in OGG1 and XRCC1 DNA repair genes...

  14. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    National Research Council Canada - National Science Library

    Goldman, Radoslav

    2006-01-01

    .... To evaluate this hypothesis, we quantify DNA repair capacity in blood cells using comet assay and evaluate how this repair capacity is related to genetic variants in OGG1 and XRCC1 DNA repair genes...

  15. DNA methylation in human fibroblasts following DNA damage and repair

    International Nuclear Information System (INIS)

    Kastan, M.B.

    1984-01-01

    Methylation of deoxycytidine (dCyd) incorporated by DNA excision repair synthesis in human diploid fibroblasts following damage with ultraviolet radiation (UV), N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene was studied utilizing [6- 3 H]dCyd to label repaired DNA specifically and high performance liquid chromatographic analysis to quantify the percentage of deoxycytidine converted to 5-methyldeoxycytidine (m 5 dCyd). In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication a level of 3.4% m 5 dCyd is reached in less than 2 hours, following UV-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approx.2.0% m 5 dCyd in the repair patch. This undermethylation of repair patches occurs throughout the genome. In cells from cultures in logarithmic-phase growth, m 5 dCyd formation in UV-induced repair patches occurs faster and to a greater extent, reaching a level of approx.2.7% in 10-20 hours. Pre-existing hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites. The distribution within chromatin of m 5 dCyd in repair patches was also investigated. Over a wide range of extents of digestion by staphylococcal nuclease or deoxyribonuclease I, the level of hypomethylation in repaired DNA in nuclease sensitive and resistant regions of chromatin was constant relative to the genomic level of methylation in these regions. Similar conclusions were reached in experiments with isolated mononucleosomes

  16. Human DNA repair and recombination genes

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs

  17. Saturation of DNA repair in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, F E; Setlow, R B

    1979-01-01

    Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20 J/m/sup 2/. We measured excision repair in normal human fibroblasts up to 80 J/m/sup 2/. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24h following two 20 J/m/sup 2/ doses separated by 24h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the rate limiting steps in repair.

  18. DNA replication and repair in Tilapia cells

    International Nuclear Information System (INIS)

    Yew, F.H.; Chang, L.M.

    1984-01-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-β-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor. (author)

  19. Laboratory of Mutagenesis and DNA Repair

    International Nuclear Information System (INIS)

    2000-01-01

    Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

  20. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...... landscape may be stably maintained even in the face of dramatic changes in chromatin structure....

  1. DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

    Science.gov (United States)

    Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori

    2016-04-02

    Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high

  2. DNA repair mechanism in radioresistant bacteria

    International Nuclear Information System (INIS)

    Kitayama, Shigeru

    1992-01-01

    Many radiation resistant bacteria have been isolated from various sources which are not in high background field. Since Deinococcus radiodurans had been isolated first in 1956, studies on the mechanism for radioresistance were carried out mostly using this bacterium. DNA in this bacterium isn't protected against injury induced by not only ionizing radiation but also ultraviolet light. Therefore, DNA damages induced by various treatments are efficiently and accurately repaired in this cells. Damages in base and/or sugar in DNA are removed by endonucleases which, if not all, are synthesized during postirradiation incubation. Following the endonucleolytic cleavage the strand scissions in DNA are seemed to be rejoined by a process common for the repair of strand scissions induced by such as ionizing radiations. Induce protein(s) is also involved in this rejoining process of strand scissions. DNA repair genes were classified into three phenotypic groups. (1)Genes which are responsible for the endonucleolytic activities. (2) Genes involved in the rejoining of DNA strand scissions. (3) Genes which participate in genetic recombination and repair. Three genes belong to (1) and (2) were cloned onto approximately 1 kbp DNA fragments which base sequences have been determined. (author)

  3. DNA repair mechanism in radioresistant bacteria

    International Nuclear Information System (INIS)

    Kitayama, Shigeru

    1992-01-01

    Many radiation resistant bacteria have been isolated from various sources which are not in high background field. Since Deinococcus radiodurans had been isolated first in 1956, the studies on the mechanism of radioresistance were mostly carried out using this bacterium. DNA in this bacterium isn't protected against injury induced by not only ionizing radiation but also ultraviolet light. Therefore, DNA damages induced by various treatments are efficiently and accurately repaired in this cells. Damages in base and/or sugar in DNA are removed by endonucleases which, if not all, are synthesized during postirradiation incubation. Following the endonucleolytic cleavage the strand scissions in DNA are seemed to be rejoined by a process common for the repair of strand scissions induced by such as ionizing radiations. Induce protein(s) is also involved in this rejoining process of strand scissions. DNA repair genes were classified into three phenotypic groups. (1) Genes which are responsible for the endonucleolytic activities. (2) Genes involved in the rejoining of DNA strand scissions. (3) Genes which participate in genetic recombination and repair. Three genes belong to (1) and (2) were cloned onto approximately 1 kbp DNA fragments which base sequences have been determined. (author)

  4. 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

  5. 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.

  6. DNA repair: keeping it together

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2004-01-01

    A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....

  7. A model system for DNA repair studies

    International Nuclear Information System (INIS)

    Lange, C.S.; Perlmutter, E.

    1984-01-01

    The search for the ''lethal lesion:'' which would yield a molecular explanation of biological survival curves led to attempts to correlate unrepaired DNA lesions with loss of reproductive integrity. Such studies have shown the crucial importance of DNA repair systems. The unrepaired DSB has been sought for such correlation, but in such study the DNA was too large, polydisperse, and/or structurally complex to permit precise measurement of break induction and repair. Therefore, an analog of higher order systems but with a genome of readily measurable size, is needed. Bacteriophage T4 is such an analog. Both its biological (PFU) and molecular (DNA) survival curves are exponentials. Its aerobic /sub PFU/D/sub 37///sub DNA/D/sub 37/ ratio, (410 +- 4.5Gy/540 +- 25 Gy) indicates that 76 +- 4% of lethality at low multiplicity infection (moi 1) the survival is greater than can be explained if the assumption of no parental DSB repair were valid. Both T4 and its host have DSB repair systems which can be studied by the infectious center method. Results of such studies are discussed

  8. Fragile DNA Repair Mechanism Reduces Ageing in Multicellular Model

    DEFF Research Database (Denmark)

    Bendtsen, Kristian Moss; Juul, Jeppe Søgaard; Trusina, Ala

    2012-01-01

    increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected...... to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity......DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn...

  9. Ancient bacteria show evidence of DNA repair

    DEFF Research Database (Denmark)

    Johnson, Sarah Stewart; Hebsgaard, Martin B; Christensen, Torben R

    2007-01-01

    -term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence...... geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long...... that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability....

  10. Modes of DNA repair and replication

    International Nuclear Information System (INIS)

    Hanawalt, P.; Kondo, S.

    1979-01-01

    Modes of DNA repair and replication require close coordination as well as some overlap of enzyme functions. Some classes of recovery deficient mutants may have defects in replication rather than repair modes. Lesions such as the pyrimidine dimers produced by ultraviolet light irradiation are the blocks to normal DNA replication in vivo and in vitro. The DNA synthesis by the DNA polymerase 1 of E. coli is blocked at one nucleotide away from the dimerized pyrimidines in template strands. Thus, some DNA polymerases seem to be unable to incorporate nucleotides opposite to the non-pairing lesions in template DNA strands. The lesions in template DNA strands may block the sequential addition of nucleotides in the synthesis of daughter strands. Normal replication utilizes a constitutive ''error-free'' mode that copies DNA templates with high fidelity, but which may be totally blocked at a lesion that obscures the appropriate base pairing specificity. It might be expected that modified replication system exhibits generally high error frequency. The error rate of DNA polymerases may be controlled by the degree of phosphorylation of the enzyme. Inducible SOS system is controlled by recA genes that also control the pathways for recombination. It is possible that SOS system involves some process other than the modification of a blocked replication apparatus to permit error-prone transdimer synthesis. (Yamashita, S.)

  11. The relationship of transcription and repair of radioinduced DNA damage

    International Nuclear Information System (INIS)

    Zhestyanikov, V.D.; Igusheva, O.A.

    1997-01-01

    The data are discussed which has become a basement of such important findings as involvement of transcription into repair or existence of transcription-coupling repair factors. Thymine glycols which are appear under ionizing radiation exposure, are repaired preferentially in transcribed DNA. In present review the preferential repair of ionizing radiation-induced singlestrand breaks (SSBa) in transcribed DNA of human cells. Discontinuous distribution of DNA repair along hole genome has a grate role in biological processes

  12. Mutagenic DNA repair in Escherichia coli. VII

    International Nuclear Information System (INIS)

    Bridges, B.A.; Mottershead, R.P.

    1978-01-01

    Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp + mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating 'fixation') during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair. In contrast the frequency of UV-induced mutations to Trp + (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. It is concluded that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system. (Auth.)

  13. Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells1

    OpenAIRE

    Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

    2004-01-01

    Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse LβT2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regu...

  14. Studies on DNA repair in Bacillus subtilis

    International Nuclear Information System (INIS)

    Inoue, Tadashi; Kada, Tsuneo

    1977-01-01

    An enzyme which enhances the priming activity of γ-irradiated DNA for type I DNA polymerase (EC 2.7.7.7) was identified and partially purified from extracts of Bacillus subtilis cells. The enzyme preferentially degraded γ-irradiated DNA into acid-soluble materials. DNA preparations treated with heat, ultraviolet light, pancreatic DNAase (EC 3.1.4.5) or micrococcal DNAase (EC 3.1.4.7) were not susceptible to the enzyme. However, sonication rendered DNA susceptible to the enzyme to some extent. From these results, it is supposed that this enzyme may function by 'cleaning' damaged terminals produced by γ-irradiation to serve as effective primer of sites for repair synthesis by the type I DNA polymerase

  15. Visualization of DNA double-strand break repair: From molecules to cells

    NARCIS (Netherlands)

    Krawczyk, Przemek M.; Stap, Jan; Aten, Jacob A.

    2008-01-01

    DNA double-strand break (DSB) signaling and repair processes are positioned at the crossroad of nuclear pathways that regulate DNA replication, cell division, senescence and apoptosis. Importantly, errors in DSB repair may lead to lethal or potentially tumorigenic chromosome rearrangements.

  16. 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...

  17. Kinetics and mechanism of DNA repair

    International Nuclear Information System (INIS)

    Meldrum, R.A.; Wharton, C.W.; Shall, S.

    1990-01-01

    Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the γ-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside trisphosphate is isotopically labelled in the α-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair. (author)

  18. RAD51 interconnects between DNA replication, DNA repair and immunity.

    Science.gov (United States)

    Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

    2017-05-05

    RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Science.gov (United States)

    2010-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  20. DNA mismatch repair, genome instability and cancer in zebrafish

    NARCIS (Netherlands)

    Feitsma, H.

    2008-01-01

    The objective of this study was to find out whether the zebrafish can be an appropriate model for studying DNA repair and cancer. For this purpose three fish lines were used that lack components of an important mechanism for the repair of small DNA damage: DNA mismatch repair. These fish are

  1. The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

    Science.gov (United States)

    Miles, Jennifer A; Frost, Mark G; Carroll, Eilis; Rowe, Michelle L; Howard, Mark J; Sidhu, Ateesh; Chaugule, Viduth K; Alpi, Arno F; Walden, Helen

    2015-08-21

    The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  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. Energy and Technology Review: Unlocking the mysteries of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  4. DNA repair in cancer: emerging targets for personalized therapy

    International Nuclear Information System (INIS)

    Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

    2014-01-01

    Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer

  5. Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

    Science.gov (United States)

    Hengel, Sarah R; Spies, M Ashley; Spies, Maria

    2017-09-21

    To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Cycling with BRCA2 from DNA repair to mitosis

    International Nuclear Information System (INIS)

    Lee, Hyunsook

    2014-01-01

    Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity. - Highlights: • BRCA2 is a multifaceted tumor suppressor and is crucial in genetic integrity. • BRCA2 exerts distinct functions in cell cycle-specific manner. • Mitotic kinases regulate diverse functions of BRCA2 in mitosis and cytokinesis

  7. Cycling with BRCA2 from DNA repair to mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyunsook, E-mail: HL212@snu.ac.kr

    2014-11-15

    Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity. - Highlights: • BRCA2 is a multifaceted tumor suppressor and is crucial in genetic integrity. • BRCA2 exerts distinct functions in cell cycle-specific manner. • Mitotic kinases regulate diverse functions of BRCA2 in mitosis and cytokinesis.

  8. Recruitment of DNA methyltransferase I to DNA repair sites

    Science.gov (United States)

    Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

    2005-01-01

    In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

  9. Stripped-down DNA repair in a highly reduced parasite

    Directory of Open Access Journals (Sweden)

    Fast Naomi M

    2007-03-01

    Full Text Available Abstract Background Encephalitozoon cuniculi is a member of a distinctive group of single-celled parasitic eukaryotes called microsporidia, which are closely related to fungi. Some of these organisms, including E. cuniculi, also have uniquely small genomes that are within the prokaryotic range. Thus, E. cuniculi has undergone a massive genome reduction which has resulted in a loss of genes from diverse biological pathways, including those that act in DNA repair. DNA repair is essential to any living cell. A loss of these mechanisms invariably results in accumulation of mutations and/or cell death. Six major pathways of DNA repair in eukaryotes include: non-homologous end joining (NHEJ, homologous recombination repair (HRR, mismatch repair (MMR, nucleotide excision repair (NER, base excision repair (BER and methyltransferase repair. DNA polymerases are also critical players in DNA repair processes. Given the close relationship between microsporidia and fungi, the repair mechanisms present in E. cuniculi were compared to those of the yeast Saccharomyces cerevisiae to ascertain how the process of genome reduction has affected the DNA repair pathways. Results E. cuniculi lacks 16 (plus another 6 potential absences of the 56 DNA repair genes sought via BLASTP and PSI-BLAST searches. Six of 14 DNA polymerases or polymerase subunits are also absent in E. cuniculi. All of these genes are relatively well conserved within eukaryotes. The absence of genes is not distributed equally among the different repair pathways; some pathways lack only one protein, while there is a striking absence of many proteins that are components of both double strand break repair pathways. All specialized repair polymerases are also absent. Conclusion Given the large number of DNA repair genes that are absent from the double strand break repair pathways, E. cuniculi is a prime candidate for the study of double strand break repair with minimal machinery. Strikingly, all of the

  10. DNA Damage Induced by Alkylating Agents and Repair Pathways

    OpenAIRE

    Natsuko Kondo; Akihisa Takahashi; Koji Ono; Takeo Ohnishi

    2010-01-01

    The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O 6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O 6-methylguanine-DNA methyltransferase, and O 6MeG:T mispairs are recognized...

  11. Investigations of DNA-repair in New Zealand mice

    Energy Technology Data Exchange (ETDEWEB)

    Tuschl, H; Kovac, R; Altmann, H

    1974-09-01

    DNA repair was investigated in New Zealand mice strains which developed murine lupus and compared with Swiss control mice. Unscheduled DNA synthesis demonstrated by autoradiography was used to measure the repair capacity of spleen cells. After gamma-irradiation DNA repair was decreased in the autoimmune strains, while it was significantly increased after UV-irradiation. A possible relationship between repair capacity after gamma-respectively UV-irradiation and the etiologic factor of autoimmunity is discussed. (auth)

  12. Role of nuclear hexokinase II in DNA repair

    International Nuclear Information System (INIS)

    Khanna, S.; Bhatt, A.N.; Dwarakanath, B.S.; Kalaiarasan, P.; Brahmachari, V.

    2012-01-01

    A common signature of many cancer cells is a high glucose catabolic rate primarily due to the over expression of Type II hexokinase (HKII; responsible for the phosphorylation of glucose), generally known as cytosolic and mitochondrial bound enzyme that also suppresses cell death. Although, nuclear localization and transcriptional regulation of HKII has been reported in yeast; we and few others have recently demonstrated its nuclear localization in malignant cell lines. Interestingly, modification of a human glioma cell line (BMG-1) for enhancing glycolysis through mitochondrial respiration (OPMBMG cells) resulted in a higher nuclear localization of HKII as compared to the parental cells with concomitant increase in DNA repair and radio-resistance. Further, the glucose phosphorylation activity of the nuclear HKII was nearly 2 folds higher in the relatively more radioresistant HeLa cells (human cervical cancer cell line) as compared to MRC-5 cells (human normal lung fibroblast cell line). Therefore, we hypothesize that nuclear HKII facilitates DNA repair, in a hither to unknown mechanism, that may partly contribute to the enhanced resistance of highly glycolytic cells to radiation. Sequence alignment studies suggest that the isoenzymes, HKI and HKII share strong homology in the kinase active site, which is also found in few protein kinases. Interestingly HKI has been shown to phosphorylate H2A in-vitro. Further, in-silico protein-protein interaction data suggest that HKII can interact with several DNA repair proteins including ATM. Taken together; available experimental evidences as well as in-silico predictions strongly suggest that HKII may play a role in DNA repair by phosphorylation of certain DNA repair proteins. (author)

  13. Repair of DNA DSB in higher eukaryotes

    International Nuclear Information System (INIS)

    Wang, H.; Perrault, A.R.; Takeda, Y.; Iliakis, G.

    2003-01-01

    Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a NHEJ apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4, and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK- dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. We studied the role of Ku and DNA-PKcs in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient error-free endjoining observed in such in-vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite that fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA endjoining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing endjoining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts sugggesting the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the

  14. Inhibition of DNA repair by trifluoperazine

    Energy Technology Data Exchange (ETDEWEB)

    Charp, P.A.; Regan, J.D.

    1985-01-01

    The authors examined the possible role of calmodulin in the excision repair of ultraviolet light-induced pyrimidine dimers in damaged DNA by means of specialized assay systems. These assays included bromodeoxyuridine photolysis, dimer chromatography and cytosine arabinoside incorporation in conjunction with hydroxyurea. The calmodulin antagonist, trifluoperazine, and the calcium-chelating agent, EGTA, were employed to ascertain what affect calmodulin played in the repair process. Normal human fibroblast cells are used in all studies described in this report. After exposure to 10 J/m2 of 254 nm light, the authors observed a decrease of about 30% in the number of single-strand breaks produced in the presence of 25 M trifluoperazine (1.9 vs. 3.3) in controls although the numbers of bases re-inserted in the repaired regions were similar (64 vs. 72). Measurement of thymine-containing dimers remaining throughout a 24 h time period indicated a 30% difference decrease in the number of cytosine arabinoside arrested repair sites in the presence of either EGTA or trifluoperazine. The results are discussed with relation to the possibility of calmodulin altering the initial incision by repair endonuclease. 27 references, 3 figures.

  15. Epigenetic changes of DNA repair genes in cancer.

    Science.gov (United States)

    Lahtz, Christoph; Pfeifer, Gerd P

    2011-02-01

    'Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

  16. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  17. DNA repair in proteus mirabilis. Pt. 4

    International Nuclear Information System (INIS)

    Hofemeister, J.

    1977-01-01

    Post-irradiation DNA degradation in P. mirabilis rec + strains after UV irradiation is found to be more extensive in starvation buffer than in growth medium. In growth medium restriction of protein synthesis, but not DNA synthesis, largely prevents the expression of 'breakdown limitation'. By the addition of chloramphenicol during post-irradiation incubation in growth medium the expression of breakdown limitation was followed and found to occur 20 to 40 min after UV irradiation. Pre-irradiation by a low dose of UV leads after a corresponding time of post-irradiation incubation to breakdown limitation even in starvation buffer after a second UV exposure. Post-irradiation DNA degradation is presumed to be initiated at the sites of DNA lesions which arise at replication points damaged by UV. While pre-starvation restricts the efficiency of postirradiation DNA degradation by the reduction of the number of replication points active at the time of irradiation, caffeine as well as 2.4-dinitrophenol inhibit DNA degradation even in rec - cells probably by the interference with nicking or exonucleoltytic events initiated at those sites in the absence of breakdown limitation. Breakdown limitation is postulated to be due to inducible derepression of REC-functions which lead to the protection and, probably, repair of DNA lesions arising at the replication points following UV exposure. (orig.) [de

  18. Spontaneous mutation by mutagenic repair of spontaneous lesions in DNA

    International Nuclear Information System (INIS)

    Hastings, P.J.; Quah, S.-K.; Borstel, R.C. von

    1976-01-01

    It is stated that strains of yeast carrying mutations in many of the steps in pathways repairing radiation-induced damage to DNA have enhanced spontaneous mutation rates. Most strains isolated because they have enhanced spontaneous mutation carry mutations in DNA repair systems. This suggests that much spontaneous mutation arises by mutagenic repair of spontaneous lesions. (author)

  19. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    Science.gov (United States)

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  20. DNA polymerase beta participates in mitochondrial DNA repair

    DEFF Research Database (Denmark)

    Sykora, P; Kanno, S; Akbari, M

    2017-01-01

    We have detected DNA polymerase beta (Polβ), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polβ in the mitochondria. Using Polβ fragments, mitocho......We have detected DNA polymerase beta (Polβ), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polβ in the mitochondria. Using Polβ fragments......, mitochondrial-specific protein partners were identified, with the interactors mainly functioning in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1 and TFAM, all of which are mitochondria specific DNA effectors and are known to function...... in the nucleoid. Polβ directly interacted with, and influenced the activity of, the mitochondrial helicase TWINKLE. Human kidney cells with Polβ knock-out (KO) had higher endogenous mtDNA damage. Mitochondrial extracts derived from heterozygous Polβ mouse tissue and KO cells had lower nucleotide incorporation...

  1. Repetitious nature of repaired DNA in mammalian cells

    International Nuclear Information System (INIS)

    1978-01-01

    The report consists of three appendices, as follows: summary of preliminary studies of the comparative DNA repair in normal lymphoblastoid and Burkitt's lymphoma cell lines; nonuniform reassociation of human lymphoblastoid cell DNA repair replicated following methyl methane sulfonate treatment; and preliminary DNA single-strand breakage studies in the L5178Y cell line

  2. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

    Energy Technology Data Exchange (ETDEWEB)

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand (Montreal)

    2010-09-07

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

  3. Histone H2AX in DNA repair

    International Nuclear Information System (INIS)

    Lewandowska, H.; Szumiel, I.

    2002-01-01

    The paper reviews the recent reports on the role of the phosphorylated histone H2AX (γ-H2AX). The modification of this histone is an important part of the cellular response to the induction of DNA double strand brakes (DSB) by ionising radiation and other DSB-generating factors. In irradiated cells the modification is carried out mainly by ATM (ataxia-telangiectasia mutated) kinase, the enzyme that starts the alarm signalling upon induction of DSB.γ-H2AX molecules are formed within 1-3 min after irradiation and form foci at the sites of DSB. This seems to be necessary for the recruitment of repair factors that are later present in foci of damaged nuclei. Modification of a constant percentage of H2AX molecules per DSB takes place, corresponding to chromatin domains of megabase of DNA. (author)

  4. 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.

  5. Mitochondrial DNA repair and association with aging- an update

    DEFF Research Database (Denmark)

    Diaz, Ricardo Gredilla; Bohr, Vilhelm; Stevnsner, Tinna V.

    2010-01-01

    in the aging process and to be particularly deleterious in post-mitotic cells. Thus, DNA repair is an important mechanism for maintenance of genomic integrity. Despite the importance of mitochondria in the aging process, it was thought for many years that mitochondria lacked an enzymatic DNA repair system...... proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process....

  6. DNA repair and radiation sensitivity in mammalian cells

    International Nuclear Information System (INIS)

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

  7. A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

    Science.gov (United States)

    Friedberg, Errol C

    2016-01-01

    This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases. Copyright © 2015. Published by Elsevier B.V.

  8. 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

  9. 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.

  10. Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ikai, K.; Tano, K.; Ohnishi, T.; Nozu, K.

    1985-01-01

    The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

  11. DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Ford, M.D.; Houldsworth, J.; Lavin, M.F. (Queensland Univ., Brisbane (Australia). Dept. of Biochemistry)

    1981-12-01

    The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair ..gamma..-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to ..gamma..-radiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrated to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10-40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.

  12. Epigenetic changes of DNA repair genes in cancer

    OpenAIRE

    Lahtz, Christoph; Pfeifer, Gerd P.

    2011-01-01

    ‘Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, ...

  13. Discovery of DNA repair inhibitors by combinatorial library profiling

    Science.gov (United States)

    Moeller, Benjamin J.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2011-01-01

    Small molecule inhibitors of DNA repair are emerging as potent and selective anti-cancer therapies, but the sheer magnitude of the protein networks involved in DNA repair processes poses obstacles to discovery of effective candidate drugs. To address this challenge, we used a subtractive combinatorial selection approach to identify a panel of peptide ligands that bind DNA repair complexes. Supporting the concept that these ligands have therapeutic potential, we show that one selected peptide specifically binds and non-competitively inactivates DNA-PKcs, a protein kinase critical in double-strand DNA break repair. In doing so, this ligand sensitizes BRCA-deficient tumor cells to genotoxic therapy. Our findings establish a platform for large-scale parallel screening for ligand-directed DNA repair inhibitors, with immediate applicability to cancer therapy. PMID:21343400

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

    Directory of Open Access Journals (Sweden)

    Andrea Pagano

    2017-11-01

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

  15. DNA repair in proteus mirabilis. 5

    International Nuclear Information System (INIS)

    Hofemeister, J.; Boehme, H.; Fleischhacker, M.; Adler, B.; Eitner, G.; Steinborn, G.

    1979-01-01

    Inducible cell lysis in UV-irradiated cultures of Proteus mirabilis PG VI rec + derivatives is due to the induction of a defective prophage dP VI. This lytic response is found to account for the extraordinary high UV sensitivity of P. mirabilis rec + strains. Lysis of cells after UV or mitomycin C treatment is accompanied by the release of various defective phage particles including head-tail, tail and polysheath structures. Survival of P. mirabilis is shown to be strongly affected by the plating procedure. It is supposed that plating of UV-irradiated rec + cells after a distinct time of growth in liquid medium some how enhances the efficiency of repair activities rather than to prevent inducible cell lysis in P. mirabilis from occurring. The resident defective prophage interferes with the multiplication of infectious temperate phages (π1) as indicated by the efficiency of plating and plaque size. The extent of host-controlled reactivation of UV-irradiated phages, however, was not influenced by the lysogenic state of the host bacteria. The derepression of the resident prophage is thought to correlate with the appearence after UV exposure of a distinct and relatively stable low molecular weight DNA which is assumed to originate from enzymatic fragmentation rather than postmortal nicking of chromosomal DNA in P. mirabilis. Mutants are described which lost the inducible cell lysis response and, in case of PG 1300- also lost the immunity for bacteriolytic activities formed by PG VI strains after lysogenic induction. Whilst this derivative of P. mirabilis might either be cured for the defective prophage or rendered noninducible by mutation this substrain lost also both the extrasensitivity against UV and the chromosomal DNA fragmentation. The extent of the UV-induced DNA degradation response seems not to be markedly affected by these DNA fragmentations. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1978-12-01

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

  17. Caffeine, cyclic AMP and postreplication repair of mammalian DNA

    International Nuclear Information System (INIS)

    Ehmann, U.K.

    1976-01-01

    The methylxanthines, caffeine and theophylline, inhibit postreplication repair of DNA in mammalian cells. Because they also inhibit cyclic AMP phosphodiesterase, it was thought that there might be some connection between concentrations of cyclic AMP and postreplication repair. This possibility was tested by performing DNA sedimentation experiments with a cyclic AMP-resistant mouse lymphoma cell mutant and its wild-type counterpart. The results show that there is no connection between cellular cyclic AMP concentrations and the rate of postreplication repair. Therefore, it is more likely that caffeine and theophylline inhibit postreplication repair by some other means, such as by binding to DNA

  18. DNA repair systems as targets of cadmium toxicity

    International Nuclear Information System (INIS)

    Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios

    2006-01-01

    Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity

  19. Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair

    International Nuclear Information System (INIS)

    Thomas, S.M.; Sedgwick, S.G.

    1989-01-01

    Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli

  20. DNA repair mechanisms in cancer development and therapy.

    Science.gov (United States)

    Torgovnick, Alessandro; Schumacher, Björn

    2015-01-01

    DNA damage has been long recognized as causal factor for cancer development. When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumor suppressor genes, cells undergo malignant transformation resulting in cancerous growth. Genetic defects can predispose to cancer: mutations in distinct DNA repair systems elevate the susceptibility to various cancer types. However, DNA damage not only comprises a root cause for cancer development but also continues to provide an important avenue for chemo- and radiotherapy. Since the beginning of cancer therapy, genotoxic agents that trigger DNA damage checkpoints have been applied to halt the growth and trigger the apoptotic demise of cancer cells. We provide an overview about the involvement of DNA repair systems in cancer prevention and the classes of genotoxins that are commonly used for the treatment of cancer. A better understanding of the roles and interactions of the highly complex DNA repair machineries will lead to important improvements in cancer therapy.

  1. DNA Repair Mechanisms in Cancer Development and Therapy

    Directory of Open Access Journals (Sweden)

    Alessandro eTorgovnick

    2015-04-01

    Full Text Available DNA damage has been long recognized as causal factor for cancer development. When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumor suppressor genes, cells undergo malignant transformation resulting in cancerous growth. Genetic defects can predispose to cancer: Mutations in distinct DNA repair systems elevate the susceptibility to various cancer types. However, DNA damage not only comprises a root cause for cancer development but also continues to provide an important avenue for chemo- and radiotherapy. Since the beginning of cancer therapy, genotoxic agents have been applied that trigger DNA damage checkpoints that halt the growth and trigger the apoptotic demise of cancer cells. We provide an overview about the involvement of DNA repair systems in cancer prevention and the classes of genotoxins that are commonly used for the treatment of cancer. A better understanding of the roles and interactions of the highly complex DNA repair machineries will lead to important improvements in cancer therapy.

  2. Rad52 SUMOylation affects the efficiency of the DNA repair

    DEFF Research Database (Denmark)

    Altmannova, Veronika; Eckert-Boulet, Nadine; Arneric, Milica

    2010-01-01

    Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification by ...

  3. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    International Nuclear Information System (INIS)

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

  4. DNA repair in neurons: So if they don't divide what's to repair?

    International Nuclear Information System (INIS)

    Fishel, Melissa L.; Vasko, Michael R.; Kelley, Mark R.

    2007-01-01

    Neuronal DNA repair remains one of the most exciting areas for investigation, particularly as a means to compare the DNA repair response in mitotic (cancer) vs. post-mitotic (neuronal) cells. In addition, the role of DNA repair in neuronal cell survival and response to aging and environmental insults is of particular interest. DNA damage caused by reactive oxygen species (ROS) such as generated by mitochondrial respiration includes altered bases, abasic sites, and single- and double-strand breaks which can be prevented by the DNA base excision repair (BER) pathway. Oxidative stress accumulates in the DNA of the human brain over time especially in the mitochondrial DNA (mtDNA) and is proposed to play a critical role in aging and in the pathogenesis of several neurological disorders including Parkinson's disease, ALS, and Alzheimer's diseases. Because DNA damage accumulates in the mtDNA more than nuclear DNA, there is increased interest in DNA repair pathways and the consequence of DNA damage in the mitochondria of neurons. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the BER pathway. Following the notion that the bulk of neuronal DNA damage is acquired by oxidative DNA damage and ROS, the BER pathway is a likely area of focus for neuronal studies of DNA repair. BER variations in brain aging and pathology in various brain regions and tissues are presented. Therefore, the BER pathway is discussed in greater detail in this review than other repair pathways. Other repair pathways including direct reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination and non-homologous end joining are also discussed. Finally, there is a growing interest in the role that DNA repair pathways play in the clinical arena as they relate to the neurotoxicity and neuropathy associated with cancer treatments. Among the numerous side effects of cancer treatments, major clinical effects

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

    Directory of Open Access Journals (Sweden)

    Annemarie Grindel

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

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

    International Nuclear Information System (INIS)

    Hanawalt, P.C.

    1987-09-01

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

  7. 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

  8. Emerging connection between centrosome and DNA repair machinery

    International Nuclear Information System (INIS)

    Shimada, Mikio; Komatsu, Kenshi

    2009-01-01

    Centrosomes function in proper cell division in animal cells. The centrosome consists of a pair of centrioles and the surrounding pericentriolar matrix (PCM). After cytokinesis, daughter cells each acquire one centrosome, which subsequently duplicates at the G1/S phase in a manner that is dependent upon CDK2/cyclin-E activity. Defects in the regulation of centrosome duplication lead to tumorigenesis through abnormal cell division and resulting inappropriate chromosome segregation. Therefore, maintenance of accurate centrosome number is important for cell fate. Excess number of centrosomes can be induced by several factors including ionizing radiation (IR). Recent studies have shown that several DNA repair proteins localize to the centrosome and are involved in the regulation of centrosome number possibly through cell cycle checkpoints or direct modification of centrosome proteins. Furthermore, it has been reported that the development of microcephaly is likely caused by defective expression of centrosome proteins, such as ASPM, which are also involved in the response to IR. The present review highlights centrosome duplication in association with genotoxic stresses and the regulatory mechanism mediated by DNA repair proteins. (author)

  9. Repair of DNA-polypeptide crosslinks by human excision nuclease

    Science.gov (United States)

    Reardon, Joyce T.; Sancar, Aziz

    2006-03-01

    DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

  10. DNA Damage Induced by Alkylating Agents and Repair Pathways

    Science.gov (United States)

    Kondo, Natsuko; Takahashi, Akihisa; Ono, Koji; Ohnishi, Takeo

    2010-01-01

    The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine. PMID:21113301

  11. Mediator MED23 Links Pigmentation and DNA Repair through the Transcription Factor MITF.

    Science.gov (United States)

    Xia, Min; Chen, Kun; Yao, Xiao; Xu, Yichi; Yao, Jiaying; Yan, Jun; Shao, Zhen; Wang, Gang

    2017-08-22

    DNA repair is related to many physiological and pathological processes, including pigmentation. Little is known about the role of the transcriptional cofactor Mediator complex in DNA repair and pigmentation. Here, we demonstrate that Mediator MED23 plays an important role in coupling UV-induced DNA repair to pigmentation. The loss of Med23 specifically impairs the pigmentation process in melanocyte-lineage cells and in zebrafish. Med23 deficiency leads to enhanced nucleotide excision repair (NER) and less DNA damage following UV radiation because of the enhanced expression and recruitment of NER factors to chromatin for genomic stability. Integrative analyses of melanoma cells reveal that MED23 controls the expression of a melanocyte master regulator, Mitf, by modulating its distal enhancer activity, leading to opposing effects on pigmentation and DNA repair. Collectively, the Mediator MED23/MITF axis connects DNA repair to pigmentation, thus providing molecular insights into the DNA damage response and skin-related diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Fanconi anemia (cross)linked to DNA repair.

    Science.gov (United States)

    Niedernhofer, Laura J; Lalai, Astrid S; Hoeijmakers, Jan H J

    2005-12-29

    Fanconi anemia is characterized by hypersensitivity to DNA interstrand crosslinks (ICLs) and susceptibility to tumor formation. Despite the identification of numerous Fanconi anemia (FANC) genes, the mechanism by which proteins encoded by these genes protect a cell from DNA interstrand crosslinks remains unclear. The recent discovery of two DNA helicases that, when defective, cause Fanconi anemia tips the balance in favor of the direct involvement of the FANC proteins in DNA repair and the bypass of DNA lesions.

  13. Exonuclease 1 and its versatile roles in DNA repair

    DEFF Research Database (Denmark)

    Keijzers, Guido; Liu, Dekang; Rasmussen, Lene Juel

    2016-01-01

    Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin...... maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions...

  14. Increased DNA-repair in spleen cells of M. Hodgkin

    International Nuclear Information System (INIS)

    Frischauf, H.; Neumann, E.; Howanietz, L.; Dolejs, I.; Tuschl, H.; Altmann, H.

    1974-11-01

    In spleen cells of control patients and cells of Morbus Hodgkin, DNA-repair after gamma- and UV-irradiation was determined measuring the incorporated 3H-thymidine activity in the DNA. Additionally, the ratio of labeled cells compared to non-labeled cells and the grains per cell were evaluated by autoradiographic investigations. DNA-content per cell was measured using pulsecytophotometry. A significant increase of DNA-repair capacity after gamma-irradiation was found by density gradient centrifugation in alkaline sucrose. The same trend could be shown by investigations of unscheduled DNA-synthesis using autoradiographic method. (author)

  15. The effect of low radiation doses on DNA repair processes

    International Nuclear Information System (INIS)

    Tuschl, H.

    1978-08-01

    Error free DNA repair processes are an important preprequisite for the maintenance of genetic integrity of cells. They are of special importance for persons therapeutically or occupationally exposed to radiation. Therefore the effect of radiation therapy and elevated natural background radiation on unscheduled DNA synthesis was tested in peripheral lymphocytes of exposed persons. Both, autoradiographic studies of unscheduled DNA synthesis and measurement of 3 H-thymidine uptake into double stranded and single-strand containing DNA fractions revealed an increase of capacity for DNA repair. (author)

  16. Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis

    International Nuclear Information System (INIS)

    Radman, M.

    1974-01-01

    A hypothesis is proposed according to which E. coli possesses an inducible DNA repair system. This hypothetical repair, which we call SOS repair, is manifested only following damage to DNA, and requires de novo protein synthesis. SOS repair in E. coli requires some known genetic elements: recA + , lex + and probably zab + . Mutagenesis by ultraviolet light is observed only under conditions of functional SOS repair: we therefore suspect that this is a mutation-prone repair. A number of phenomena and experiments is reviewed which at this point can best be interpreted in terms of an inducible mutagenic DNA repair system. Two recently discovered phenomena support the proposed hypothesis: existence of a mutant (tif) which, after a shift to elevated temperature, mimicks the effect of uv irradiation in regard to repair of phage lambda and uv mutagenesis, apparent activation of SOS repair by introduction into the recipient cell of damaged plasmid or Hfr DNA. Several specific predictions based on SOS repair hypothesis are presented in order to stimulate further experimental tests. (U.S.)

  17. Situation-dependent repair of DNA damage in yeast

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  18. Investigation of DNA damage and repair mechanism using deinococcus radiodurans

    International Nuclear Information System (INIS)

    Lau How Mooi; Kikuchi, M.; Kobayashi, Y.; Narumi, I.; Watanabe, H.

    1997-01-01

    Deninococcus Radiodurans, formerly known as Micrococcus Radiodurans, is a popular bacterium because of its high resistance to damage by carcinogens such as ionizing radiation (Dean et. al. 1966; Kitayama and Matsuyama 1968) and UV radiation (Gasvon et. al., 1995; Arrange et. al. 1993). In this report, we investigated the high resistance to ionizing radiation by this bacterium. The bacteria had been exposed from I to 5 kGy of gamma radiation and then incubated in TGY medium to study their ability to repair the broken DNA. The repair time was measured by Pulse Field Gel Electrophoresis (PFGE) method. The repair time for each dose was determined. Also in order to ensure that the repair was perfect, the bacterium was subjected to a second exposure of ionizing radiation after it has fully repaired. It was found that the 'second' repair characteristic was similar to the first repair. This confirmed that the repair after the exposure to the ionizing radiation was perfect

  19. Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages.

    Science.gov (United States)

    Arcanjo, Caroline; Armant, Olivier; Floriani, Magali; Cavalie, Isabelle; Camilleri, Virginie; Simon, Olivier; Orjollet, Daniel; Adam-Guillermin, Christelle; Gagnaire, Béatrice

    2018-04-27

    Tritium ( 3 H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H 2 O 2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. DNA repair in ultraviolet-irradiated spores of Bacillus subtilis

    International Nuclear Information System (INIS)

    Wang, T.C.V.

    1976-01-01

    It has been shown previously by others that at least two independent repair mechanisms are present in Bacillus subtilis for removing ''spore photoproduct'' from DNA of ultraviolet (254 nm)-irradiated spores after germination. One of these, designated as ''spore repair,'' is shown in this study to restore ''spore photoproduct'' to two thymine residues, leaving the DNA backbone intact at the end of the process in vivo. The circumstances under which this repair can occur and some characteristics of its energy requirements have been clarified. The second repair process is identified as excision repair, which can excise both ''spore photoproduct'' from DNA of irradiated spores and cyclobutane-type pyrimidine dimers from DNA of irradiated vegetative cells. In this study it is shown that the gene hcr 1 affects an enzyme activity for the incision step initiating this repair, while the gene hcr 42 affects a step subsequent to incision in the mechanism. In addition a third, independent repair system, termed ''germinative excision repair,'' is discovered and shown to be specific for excising only cyclobutane-type pyrimidine dimers but not ''spore photoproduct.'' This repair system is responsible for the observed high ultraviolet-resistance and temporary capacity for host cell reactivation on recently germinated spores of Bacillus subtilis HCR - strains

  1. 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.

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

    Science.gov (United States)

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

    2010-09-01

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

  3. Induced DNA repair pathway in mammalian cells

    International Nuclear Information System (INIS)

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 μM cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells

  4. [Biomarkers of radiation-induced DNA repair processes].

    Science.gov (United States)

    Vallard, Alexis; Rancoule, Chloé; Guy, Jean-Baptiste; Espenel, Sophie; Sauvaigo, Sylvie; Rodriguez-Lafrasse, Claire; Magné, Nicolas

    2017-11-01

    The identification of DNA repair biomarkers is of paramount importance. Indeed, it is the first step in the process of modulating radiosensitivity and radioresistance. Unlike tools of detection and measurement of DNA damage, DNA repair biomarkers highlight the variations of DNA damage responses, depending on the dose and the dose rate. The aim of the present review is to describe the main biomarkers of radiation-induced DNA repair. We will focus on double strand breaks (DSB), because of their major role in radiation-induced cell death. The most important DNA repair biomarkers are DNA damage signaling proteins, with ATM, DNA-PKcs, 53BP1 and γ-H2AX. They can be analyzed either using immunostaining, or using lived cell imaging. However, to date, these techniques are still time and money consuming. The development of "omics" technologies should lead the way to new (and usable in daily routine) DNA repair biomarkers. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

  5. 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.)

  6. Differential recruitment of DNA Ligase I and III to DNA repair sites

    Science.gov (United States)

    Mortusewicz, Oliver; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

    2006-01-01

    DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair. PMID:16855289

  7. Targeting abnormal DNA double strand break repair in cancer

    OpenAIRE

    Rassool, Feyruz V.; Tomkinson, Alan E.

    2010-01-01

    A major challenge in cancer treatment is the development of therapies that target cancer cells with little or no toxicity to normal tissues and cells. Alterations in DNA double strand break (DSB) repair in cancer cells include both elevated and reduced levels of key repair proteins and changes in the relative contributions of the various DSB repair pathways. These differences can result in increased sensitivity to DSB-inducing agents and increased genomic instability. The development of agent...

  8. DNA repair processes and their impairment in some human diseases

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1977-01-01

    Some human diseases show enhanced sensitivity to the action of environmental mutagens, and among these several are known which are defective in the repair of damaged DNA. Xeroderma pigmentosum (XP) is mainly defective in excision repair of a large variety of damaged DNA bases caused by ultraviolet light and chemical mutagens. XP involves at least 6 distinct groups, some of which may lack cofactors required for excising damage from chromatin. As a result of these defects the sensitivity of XP cells to many mutagens is increased 5- to 10-fold. Ataxia telangiectasia and Fanconi's anemia may similarly involve defects in repair of certain DNA base damage or cross-links, respectively. But most of these and other mutagen-sensitive diseases only show increases of about 2-fold in sensitivity to mutagens, and the biochemical defects in the diseases may be more complex and less directly involved in DNA repair than in XP. (Auth.)

  9. Molecular mechanisms of DNA repair inhibition by caffeine

    Energy Technology Data Exchange (ETDEWEB)

    Selby, C.P.; Sancar, A. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))

    1990-05-01

    Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  11. Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

    Science.gov (United States)

    2012-10-11

    Rev Mol Cell Biol 7: 335–346. 7. Li GM (2008) Mechanisms and functions of DNA mismatch repair. Cell Res 18: 85–98. 8. Pavlov YI, Mian IM, Kunkel TA...11: 165–170. 41. Li F, Tian L, Gu L, Li GM (2009) Evidence that nucleosomes inhibit mismatch repair in eukaryotic cells. J Biol Chem 284: 33056–33061

  12. Alkylation Induced DNA Repair and Mutagenesis in Escherichia coli.

    Science.gov (United States)

    1987-11-23

    unrepaired 3-methyladenine in DNA 29 2.4.1 Cytotoxic effects of persisting m3A in DNA 30 2.4.2 Mutagenic bypass synthesis of depurinat ,d DNA 30 3 CONCLUDING...induced by a single exposure to the ca’rcinogen N- methyl-N- nitrosourea (MNU) due to activation of the malignant Ha-ras-i locus. Analysis of the induced...ing CO:A uolymerase I for repair synthesis . Since DNA polymerase I would be required to complete repair after the in~uial activity of TagII, we tested

  13. Cranial CT and MRI in diseases with DNA repair defects

    International Nuclear Information System (INIS)

    Demaerel, P.; Kendall, B.E.; Kingsley, D.

    1992-01-01

    The CT and MRI appearances of 5 patients with Cockayne's syndrome, 5 with ataxia telangiectasia and 1 with Fanconi's anaemia are reported. These conditions, together with Bloom's syndrome and xeroderma pigmentosum are regarded as disorders of DNA repair. Characteristic CT and MRI features of Cockayne's syndrome include generalised atrophy, calcification in basal ganglia and dentate nuclei and white matter low density. Neuroradiological findings in the other DNA repair disorders are nonspecific. (orig.)

  14. The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

    Science.gov (United States)

    Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

    2014-07-01

    To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure

  15. 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.

  16. 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.

  17. Xeroderma Pigmentosum: defective DNA repair causes skin cancer and neurodegeneration

    International Nuclear Information System (INIS)

    Robbins, J.H.

    1988-01-01

    Xeroderma pigmentosum is a rare autosomal recessive disease with numerous malignancies on sun-exposed areas of the skin and eye because of an inability to repair DNA damage inflicted by harmful ultraviolet (UV) radiation of the sun. Because it is the only disease in which cancer is known to result from defective DNA repair, XP has received intense clinical and biochemical study during the last two decades. Furthermore, some patients with XP develop a primary neuronal degeneration, probably due to the inability of nerve cells to repair damage to their DNA caused by intraneuronal metabolites and physicochemical events that mimic the effects of UV radiation. Studies of XP neurodegeneration and DNA-repair defects have led to the conclusion that efficient DNA repair is required to prevent premature death of human nerve cells. Since XP neurodegeneration has similarities to premature death of nerve cells that occurs in such neurodegenerative disorders, XP may be the prototype for these more common neurodegenerations. Recent studies indicate that these degenerations also may have DNA-repair defects

  18. ErbB2 regulates NHEJ repair pathway by affecting erbB1-triggered IR-induced Akt activity

    International Nuclear Information System (INIS)

    Toulany, Mahmoud; Peter Rodemann, H.

    2009-01-01

    We have already reported that erbBl-PI3K-AKT signaling is an important pathway in regulating radiation sensitivity and DNA double strand break repair of human tumor cells. In the present study using small interfering RNA and pharmacological inhibitors in non-small cell lung cancer cell lines we investigated the role of Aktl on radiation-induced DNA-PKcs activity and DNA-double strand break (DNA-DSB) repair. Likewise, the function of erbB2 as hetrodimerization partner of erbBl in radiation-induced Akt activity and regulation of DNA-dsb repair through DNA-PKcs was evaluated. In A549 and H460 transfected with AKTl-siRNA radiation-induced phosphorylation of DNA-PKcs the key enzyme regulating NHEJ repair pathway was markedly inhibited. In both cell lines downregulation of Aktl led to a significant enhancement of residual DNA-DSB, i.e. impaired DNA-DSB repair. Interestingly, in cells transfected with DNA-PKcs-siRNA a lack of effect of AKTl-siRNA on enhancement of residual DNA-DSBs was observed. This results indicate that Aktl regulates NHEJ repair in a DNA-PKcs dependent manner

  19. Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

    International Nuclear Information System (INIS)

    Raffin, A.L.

    2009-06-01

    DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

  20. Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients with Gastric Cancer

    Science.gov (United States)

    Sahasrabudhe, Ruta; Lott, Paul; Bohorquez, Mabel; Toal, Ted; Estrada, Ana P.; Suarez, John J.; Brea-Fernández, Alejandro; Cameselle-Teijeiro, José; Pinto, Carla; Ramos, Irma; Mantilla, Alejandra; Prieto, Rodrigo; Corvalan, Alejandro; Norero, Enrique; Alvarez, Carolina; Tapia, Teresa; Carvallo, Pilar; Gonzalez, Luz M.; Cock-Rada, Alicia; Solano, Angela; Neffa, Florencia; Valle, Adriana Della; Yau, Chris; Soares, Gabriela; Borowsky, Alexander; Hu, Nan; He, Li-Ji; Han, Xiao-You; Taylor, Philip R.; Goldstein, Alisa M.; Torres, Javier; Echeverry, Magdalena; Ruiz-Ponte, Clara; Teixeira, Manuel R.; Carvajal Carmona, Luis G.

    2016-01-01

    Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer. PMID:28024868

  1. The time course of repair of ultraviolet-induced DNA damage; implications for the structural organization of repair

    International Nuclear Information System (INIS)

    Collins, A.; Squires, S.

    1986-01-01

    Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the 'random collision' model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a 'processive' mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate until scanning is complete. The authors have examined the time course of DNA repair in human fibroblasts given low doses of UV light. Using 3 distinct assays, the authors find no sign of a constant repair rate after 4 J/m 2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. (Auth.)

  2. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K., E-mail: sskumar@barc.gov.in

    2015-09-15

    Highlights: • Nrf2 inhibition in A549 cells led to attenuated DNA repair and radiosensitization. • Influence of Nrf2 on DNA repair is not linked to its antioxidant function. • Nrf2 influences DNA repair through homologous recombination (HR) repair pathway. • Many genes involved in HR pathway show ARE sequences in their upstream region. - Abstract: Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2

  3. DNA repair and its coupling to DNA replication in eukaryotic cells. [UV, x ray

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.

    1978-01-01

    This review article with 184 references presents the view that mammalian cells have one major repair system, excision repair, with many branches (nucleotide excision repair, base excision repair, crosslink repair, etc.) and a multiplicity of enzymes. Any particular carcinogen makes a spectrum of damaged sites and each kind of damage may be repaired by one or more branches of excision repair. Excision repair is rarely complete, except at very low doses, and eukaryotic cells survive and replicate DNA despite the presence of unrepaired damage. An alteration in a specific biochemical pathway seen in damaged or mutant cells will not always be the primary consequence of damage or of the biochemical defect of the cells. Detailed kinetic data are required to understand comprehensively the various facets of excision repair and replication. Correlation between molecular events of repair and cytological and cellular changes such as chromosomal damage, mutagenesis, transformation, and carcinogenesis are also rudimentary.

  4. Human longevity and variation in DNA damage response and repair

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning...... in genotyping procedures and investigated SNPs, potentially inducing differences in the coverage of gene regions. Specifically, five genes were not covered at all in the German data. Therefore, investigations in additional study populations are needed before final conclusion can be drawn....

  5. DNA damage and repair in human skin in situ

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-01

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

  6. DNA damage and repair in human skin in situ

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  7. DNA repair: a changing geography? (1964-2008).

    Science.gov (United States)

    Maisonobe, Marion; Giglia-Mari, Giuseppina; Eckert, Denis

    2013-07-01

    This article aims to explain the current state of DNA Repair studies' global geography by focusing on the genesis of the community. Bibliometric data is used to localize scientific activities related to DNA Repair at the city level. The keyword "DNA Repair" was introduced first by American scientists. It started to spread after 1964 that is to say, after P. Howard-Flanders (Yale University), P. Hanawalt (Stanford University) and R. Setlow (Oak Ridge Laboratories) found evidence for Excision Repair mechanisms. It was the first stage in the emergence of an autonomous scientific community. In this article, we will try to assess to what extent the geo-history of this scientific field is determinant in understanding its current geography. In order to do so, we will localize the places where the first "DNA Repair" publications were signed fifty years ago and the following spatial diffusion process, which led to the current geography of the field. Then, we will focus on the evolution of the research activity of "early entrants" in relation to the activity of "latecomers". This article is an opportunity to share with DNA Repair scientists some research results of a dynamic field in Science studies: spatial scientometrics. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Immunohistochemical analysis of oxidative stress and DNA repair proteins in normal mammary and breast cancer tissues

    International Nuclear Information System (INIS)

    Curtis, Carol D; Thorngren, Daniel L; Nardulli, Ann M

    2010-01-01

    During the course of normal cellular metabolism, oxygen is consumed and reactive oxygen species (ROS) are produced. If not effectively dissipated, ROS can accumulate and damage resident proteins, lipids, and DNA. Enzymes involved in redox regulation and DNA repair dissipate ROS and repair the resulting damage in order to preserve a functional cellular environment. Because increased ROS accumulation and/or unrepaired DNA damage can lead to initiation and progression of cancer and we had identified a number of oxidative stress and DNA repair proteins that influence estrogen responsiveness of MCF-7 breast cancer cells, it seemed possible that these proteins might be differentially expressed in normal mammary tissue, benign hyperplasia (BH), ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC). Immunohistochemistry was used to examine the expression of a number of oxidative stress proteins, DNA repair proteins, and damage markers in 60 human mammary tissues which were classified as BH, DCIS or IBC. The relative mean intensity was determined for each tissue section and ANOVA was used to detect statistical differences in the relative expression of BH, DCIS and IBC compared to normal mammary tissue. We found that a number of these proteins were overexpressed and that the cellular localization was altered in human breast cancer tissue. Our studies suggest that oxidative stress and DNA repair proteins not only protect normal cells from the damaging effects of ROS, but may also promote survival of mammary tumor cells

  9. DNA repair in DNA-polymerase-deficient mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Smith, D.W.; Tait, R.C.; Harris, A.L.

    1975-01-01

    Escherichia coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III can efficiently and completely execute excision-repair and postreplication repair of the uv-damaged DNA at 30 0 C and 43 0 C when assayed by alkaline sucrose gradients. Repair by Pol I - and Pol I - , Pol II - cells is inhibited by 1-β-D-arabinofuranosylcytosine (araC) at 43 0 C but not at 30 0 C, whereas that by Pol III - cells is insensitive to araC at any temperature. Thus, either Pol I or Pol III is required for complete and efficient repair, and in their absence Pol II mediates a limited, incomplete dark repair of uv-damaged DNA

  10. DNA repair is indispensable for survival after acute inflammation

    Science.gov (United States)

    Calvo, Jennifer A.; Meira, Lisiane B.; Lee, Chun-Yue I.; Moroski-Erkul, Catherine A.; Abolhassani, Nona; Taghizadeh, Koli; Eichinger, Lindsey W.; Muthupalani, Sureshkumar; Nordstrand, Line M.; Klungland, Arne; Samson, Leona D.

    2012-01-01

    More than 15% of cancer deaths worldwide are associated with underlying infections or inflammatory conditions, therefore understanding how inflammation contributes to cancer etiology is important for both cancer prevention and treatment. Inflamed tissues are known to harbor elevated etheno-base (ε-base) DNA lesions induced by the lipid peroxidation that is stimulated by reactive oxygen and nitrogen species (RONS) released from activated neutrophils and macrophages. Inflammation contributes to carcinogenesis in part via RONS-induced cytotoxic and mutagenic DNA lesions, including ε-base lesions. The mouse alkyl adenine DNA glycosylase (AAG, also known as MPG) recognizes such base lesions, thus protecting against inflammation-associated colon cancer. Two other DNA repair enzymes are known to repair ε-base lesions, namely ALKBH2 and ALKBH3; thus, we sought to determine whether these DNA dioxygenase enzymes could protect against chronic inflammation-mediated colon carcinogenesis. Using established chemically induced colitis and colon cancer models in mice, we show here that ALKBH2 and ALKBH3 provide cancer protection similar to that of the DNA glycosylase AAG. Moreover, Alkbh2 and Alkbh3 each display apparent epistasis with Aag. Surprisingly, deficiency in all 3 DNA repair enzymes confers a massively synergistic phenotype, such that animals lacking all 3 DNA repair enzymes cannot survive even a single bout of chemically induced colitis. PMID:22684101

  11. DNA-repair gene variants are associated with glioblastoma survival

    DEFF Research Database (Denmark)

    Wibom, Carl; Sjöström, Sara; Henriksson, Roger

    2012-01-01

    Abstract Patient outcome from glioma may be influenced by germline variation. Considering the importance of DNA repair in cancer biology as well as in response to treatment, we studied the relationship between 1458 SNPs, which captured the majority of the common genetic variation in 136 DNA repai...

  12. Modulation of DNA base excision repair during neuronal differentiation

    DEFF Research Database (Denmark)

    Sykora, Peter; Yang, Jenq-Lin; Ferrarelli, Leslie K

    2013-01-01

    DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

  13. At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence

    Directory of Open Access Journals (Sweden)

    Ryosuke eOhsawa

    2013-07-01

    Full Text Available It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence.

  14. Recombinant methods for screening human DNA excision repair proficiency

    International Nuclear Information System (INIS)

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

  15. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    Science.gov (United States)

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  16. DNA repair in murine embryonic stem cells and differentiated cells

    International Nuclear Information System (INIS)

    Tichy, Elisia D.; Stambrook, Peter J.

    2008-01-01

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells

  17. The role of DNA repair in herpesvirus pathogenesis.

    Science.gov (United States)

    Brown, Jay C

    2014-10-01

    In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

  18. Differences in mutagenic and recombinational DNA repair in enterobacteria

    International Nuclear Information System (INIS)

    Sedgwick, S.G.; Goodwin, P.A.

    1985-01-01

    The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed

  19. Effect of donor age on DNA repair by articular chondrocytes

    International Nuclear Information System (INIS)

    Lipman, J.M.

    1986-01-01

    The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by two different measures: unscheduled DNA synthesis (UDS) and O 6 -methylguanine acceptor protein (MGAP) activity. UDS following damage by 254 nm ultraviolet irradiation (20J/m 2 ) was examined in intact articular cartilage from rabbits of different ages. Semiconservative DNA synthesis was suppressed with hydroxurea and repair followed by the incorporation of [ 3 H]-thymidine ([ 3 H]-dThd). After repair the cartilage was digested in proteinase K (0.5mg/ml) with dodecyl sodium sulfate (0.2%) and DNA determined with Hoechst 33258 dye. UDS (dpm [ 3 H]-dThd/μg DNA) was greater in articular cartilage from 3- than 39-month-old rabbits. MGAP was studied in cell extracts of cultured human and rabbit chondrocytes by transfer of [ 3 H] O 6 -methyl groups from exogenous DNA to protein. It was significantly less in rabbit than in human cells on a per protein or DNA basis. There was no decline in this activity in human chondrocytes from newborn to 60 years of age; and rabbits from 3- to 36-months-old. The data indicate that in the two different repair mechanisms, age differences are found with resting but not dividing chondrocytes

  20. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

  1. Electron Transfer Mechanisms of DNA Repair by Photolyase

    Science.gov (United States)

    Zhong, Dongping

    2015-04-01

    Photolyase is a flavin photoenzyme that repairs two DNA base damage products induced by ultraviolet (UV) light: cyclobutane pyrimidine dimers and 6-4 photoproducts. With femtosecond spectroscopy and site-directed mutagenesis, investigators have recently made significant advances in our understanding of UV-damaged DNA repair, and the entire enzymatic dynamics can now be mapped out in real time. For dimer repair, six elementary steps have been characterized, including three electron transfer reactions and two bond-breaking processes, and their reaction times have been determined. A unique electron-tunneling pathway was identified, and the critical residues in modulating the repair function at the active site were determined. The dynamic synergy between the elementary reactions for maintaining high repair efficiency was elucidated, and the biological nature of the flavin active state was uncovered. For 6-4 photoproduct repair, a proton-coupled electron transfer repair mechanism has been revealed. The elucidation of electron transfer mechanisms and two repair photocycles is significant and provides a molecular basis for future practical applications, such as in rational drug design for curing skin cancer.

  2. Mycobacterium tuberculosis DNA repair in response to subinhibitory concentrations of ciprofloxacin.

    Science.gov (United States)

    O'Sullivan, D M; Hinds, J; Butcher, P D; Gillespie, S H; McHugh, T D

    2008-12-01

    To investigate how the SOS response, an error-prone DNA repair pathway, is expressed following subinhibitory quinolone treatment of Mycobacterium tuberculosis. Genome-wide expression profiling followed by quantitative RT (qRT)-PCR was used to study the effect of ciprofloxacin on M. tuberculosis gene expression. Microarray analysis showed that 16/110 genes involved in DNA protection, repair and recombination were up-regulated. There appeared to be a lack of downstream genes involved in the SOS response. qRT-PCR detected an induction of lexA and recA after 4 h and of dnaE2 after 24 h of subinhibitory treatment. The pattern of gene expression observed following subinhibitory quinolone treatment differed from that induced after other DNA-damaging agents (e.g. mitomycin C). The expression of the DnaE2 polymerase response was significantly delayed following subinhibitory quinolone exposure.

  3. Targeting DNA repair systems in antitubercular drug development.

    Science.gov (United States)

    Minias, Alina; Brzostek, Anna; Dziadek, Jaroslaw

    2018-01-28

    Infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, are difficult to treat using currently available chemotherapeutics. Clinicians agree on the urgent need for novel drugs to treat tuberculosis. In this mini review, we summarize data that prompts the consideration of DNA repair-associated proteins as targets for the development of new antitubercular compounds. We discuss data, including gene expression data, that highlight the importance of DNA repair genes during the pathogenic cycle as well as after exposure to antimicrobials currently in use. Specifically, we report experiments on determining the essentiality of DNA repair-related genes. We report the availability of protein crystal structures and summarize discovered protein inhibitors. Further, we describe phenotypes of available gene mutants of M. tuberculosis and model organisms Mycobacterium bovis and Mycobacterium smegmatis. We summarize experiments regarding the role of DNA repair-related proteins in pathogenesis and virulence performed both in vitro and in vivo during the infection of macrophages and animals. We detail the role of DNA repair genes in acquiring mutations, which influence the rate of drug resistance acquisition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

    Science.gov (United States)

    Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

    2007-07-01

    Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

  5. Repair of damaged DNA in-vivo. Comprehensive progress report, August 1980-August 1983

    International Nuclear Information System (INIS)

    Hanawalt, P.C.

    1983-07-01

    We have extended our characterization of long patch excision repair (LPER) and have demonstrated that LPER is not mutagenic (or error-prone); that the recA function is required for LPER, at least for its regulation; that the substrate for LPER is produced as a linear (not an exponential) function of uv (254 nm) dose; and that LPER can occur in uvr - cells treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG). We have developed 3 methods for measuring the frequency of interstrand crosslinks in DNA and are now applying these methods to the study of the formation and repair of DNA crosslinks in E.Coli. We have developed a monoclonal antibody specific for thymine glycol in DNA, and are using it to study the repair of thymine glycol in E. coli

  6. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    DEFF Research Database (Denmark)

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms...... for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance....... In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity...

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

    International Nuclear Information System (INIS)

    Keszenman-Pereyra, David

    1990-01-01

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

  8. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  9. DNA repair in lens cells during chick embryo development

    International Nuclear Information System (INIS)

    Counis, M.F.; Chaudun, E.; Simonneau, L.; Courtois, Y.

    1979-01-01

    When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenon of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [ 3 H)thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in the experimental system the synthesis of delta- and αcrystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case. (Auth.)

  10. DNA repair in mutagen-injured higher plants

    International Nuclear Information System (INIS)

    Veleminsky, J.; Gichner, T.

    1978-01-01

    Data are summarized proving the occurrence of photoreactivation of UV-induced pyrimidine dimers in cells of Nicotiana tabucum, Gingko and carrot, the excision of dimers in cells of Nicotiana tabacum, Gingko and carrot, the excision of dimers in protoplasts of carrot and in embryos of Lathyrus sativus, and the repair of DNA single-strand breaks induced in carrot protoplasts and barley embryonic cells by ionizing radiation. In irradiated barley embryos the unscheduled DNA synthesis and higher accessibility of induced primers to DNA polymerase I of E. coli were observed preferentially in G 1 cells with diffused chromatin. These reactions were inhibited by caffeine and EDTA. Unscheduled DNA synthesis was also observed in synchronized irradiated root cuttings of Vicia faba and in barley embryos treated with 4-nitroquinoline oxide, the latter being inhibited by caffeine and hydroxyurea. Repair synthesis was also established in barley embryos treated with mutagenic N-methyl-N-nitrosourea under conditions that postponed the onset of germination after the treatment. The same conditions enhanced the repair of DNA single-strand breaks induced by this mutagen and several other monofunctional alkylating compounds. From tissues of barley and of Phaseolus multiflorus, endonucleases for apurinic sites were isolated and characterized. Some of them are located in chromatin, others in chloroplasts. The relation between DNA repair and genetic effects of mutagens in higher plants is also discussed. (Auth.)

  11. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    International Nuclear Information System (INIS)

    Dupuy, Aurélie; Sarasin, Alain

    2015-01-01

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients

  12. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  13. Inducibility of error-prone DNA repair in yeast

    International Nuclear Information System (INIS)

    Siede, W.; Eckardt, F.

    1984-01-01

    Whereas some experimental evidence suggests that mutagenesis in yeast after treatment with DNA-damaging agents involves inducible functions, a general-acting error-prone repair activity analogous to the SOS system of Escherichia coli has not yet been demonstrated. The current literature on the problem of inducibility of mutagenic repair in yeast is reviewed with emphasis on the differences in the experimental procedures applied. (orig.)

  14. The effect of DNA repair defects on reproductive performance in nucleotide excision repair (NER) mouse models: an epidemiological approach

    NARCIS (Netherlands)

    Tsai, P.S.; Nielen, M.; Horst, G.T.J. van der; Colenbrander, B.; Heesterbeek, J.A.P.; Fentener van Vlissingen, J.M.

    2005-01-01

    In this study, we used an epidemiological approach to analyze an animal database of DNA repair deficient mice on reproductive performance in five Nucleotide Excision Repair (NER) mutant mouse models on a C57BL/6 genetic background, namely CSA, CSB, XPA, XPC [models for the human DNA repair disorders

  15. Stress and DNA repair biology of the Fanconi anemia pathway

    Science.gov (United States)

    Longerich, Simonne; Li, Jian; Xiong, Yong; Sung, Patrick

    2014-01-01

    Fanconi anemia (FA) represents a paradigm of rare genetic diseases, where the quest for cause and cure has led to seminal discoveries in cancer biology. Although a total of 16 FA genes have been identified thus far, the biochemical function of many of the FA proteins remains to be elucidated. FA is rare, yet the fact that 5 FA genes are in fact familial breast cancer genes and FA gene mutations are found frequently in sporadic cancers suggest wider applicability in hematopoiesis and oncology. Establishing the interaction network involving the FA proteins and their associated partners has revealed an intersection of FA with several DNA repair pathways, including homologous recombination, DNA mismatch repair, nucleotide excision repair, and translesion DNA synthesis. Importantly, recent studies have shown a major involvement of the FA pathway in the tolerance of reactive aldehydes. Moreover, despite improved outcomes in stem cell transplantation in the treatment of FA, many challenges remain in patient care. PMID:25237197

  16. Identification of DNA repair genes in the human genome

    International Nuclear Information System (INIS)

    Hoeijmakers, J.H.J.; van Duin, M.; Westerveld, A.; Yasui, A.; Bootsma, D.

    1986-01-01

    To identify human DNA repair genes we have transfected human genomic DNA ligated to a dominant marker to excision repair deficient xeroderma pigmentosum (XP) and CHO cells. This resulted in the cloning of a human gene, ERCC-1, that complements the defect of a UV- and mitomycin-C sensitive CHO mutant 43-3B. The ERCC-1 gene has a size of 15 kb, consists of 10 exons and is located in the region 19q13.2-q13.3. Its primary transcript is processed into two mRNAs by alternative splicing of an internal coding exon. One of these transcripts encodes a polypeptide of 297 aminoacids. A putative DNA binding protein domain and nuclear location signal could be identified. Significant AA-homology is found between ERCC-1 and the yeast excision repair gene RAD10. 58 references, 6 figures, 1 table

  17. Repair of uv damaged DNA in systemic lupus erythematosus. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Beighlie, D J; Teplitz, R L

    1975-06-01

    The NZB NZW hybrid mouse is an animal model of human systemic lupus erythematosus (SLE). Two breeding schemes were devised using NZB, NZW, B/W, and CBA mice, which permit definitive decisions regarding genetic and/or viral origin of the disease. It is proposed that at least two factors must be involved: a genetic abnormality producing hyper-responsiveness to nucleic acid antigens, and a DNA repair defect which results in liberation of DNA and RNA when cells are lethally injured. Evidence is presented for a DNA repair deficit in human SLE lymphocytes following in vitro irradiation with ultraviolet (uv) light. Lymphocytes from adult New Zealand and control mice were found to lack normal amounts of endonuclease necessary for repairing uv damage.

  18. Theoretical approach of complex DNA lesions: from formation to repair

    International Nuclear Information System (INIS)

    Bignon, Emmanuelle

    2017-01-01

    This thesis work is focused on the theoretical modelling of DNA damages, from formation to repair. Several projects have been led in this framework, which can be sorted into three different parts. One on hand, we studied complex DNA reactivity. It included a study about 8-oxo-7,8-dihydro-guanine (8oxoG) mechanisms of formation, a project concerning the UV-induced pyrimidine 6-4 pyrimidone (6-4PP) endogenous photo-sensitizer features, and another one about DNA photo-sensitization by nonsteroidal anti-inflammatory drugs (i.e. ketoprofen and ibuprofen). On the other hand, we investigated mechanical properties of damaged DNA. The structural signature of a DNA lesion is of major importance for their repair, unfortunately only few NMR and X-ray structures of such systems are available. In order to gain insights into their dynamical structure, we investigated a series of complex damages: clustered abasic sites, interstrand cross-links, and the 6-4PP photo-lesion. Likewise, we studied the interaction modes DNA with several polyamines, which are well known to interact with the double helix, but also with the perspective to model DNA-protein cross-linking. The third part concerned the study of DNA interactions with repair enzymes. In line with the structural study about clustered abasic sites, we investigated the dynamics of the same system, but this time interacting with the APE1 endonuclease. We also studied interactions between the Fpg glycosylase with an oligonucleotides containing tandem 8-oxoG on one hand and 8-oxoG - abasic site as multiply damaged sites. Thus, we shed new lights on damaged DNA reactivity, structure and repair, which provides perspectives for biomedicine and life's mechanisms understanding as we begin to describe nucleosomal DNA. (author)

  19. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

    Directory of Open Access Journals (Sweden)

    Elisa Mentegari

    2016-08-01

    Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

  20. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

    Science.gov (United States)

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-08-31

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

  1. Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.

    Science.gov (United States)

    Nakanishi, Koji; Yang, Yun-Gui; Pierce, Andrew J; Taniguchi, Toshiyasu; Digweed, Martin; D'Andrea, Alan D; Wang, Zhao-Qi; Jasin, Maria

    2005-01-25

    Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca-/- cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.

  2. 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)

  3. uv photobiology: DNA damage and repair

    International Nuclear Information System (INIS)

    Sutherland, B.M.

    1978-01-01

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

  4. Cranial CT and MRI in diseases with DNA repair defects

    Energy Technology Data Exchange (ETDEWEB)

    Demaerel, P.; Kendall, B.E.; Kingsley, D. (Dept. of Neuroradiology, Hospital for Sick Children, London (United Kingdom))

    1992-04-01

    The CT and MRI appearances of 5 patients with Cockayne's syndrome, 5 with ataxia telangiectasia and 1 with Fanconi's anaemia are reported. These conditions, together with Bloom's syndrome and xeroderma pigmentosum are regarded as disorders of DNA repair. Characteristic CT and MRI features of Cockayne's syndrome include generalised atrophy, calcification in basal ganglia and dentate nuclei and white matter low density. Neuroradiological findings in the other DNA repair disorders are nonspecific. (orig.).

  5. The influence of DNA repair inhibitors on the mutation rate

    International Nuclear Information System (INIS)

    Auzinger, Th.; Hruby, R.

    1980-12-01

    The simultaneous influence of gamma-radiation and DNA-repair inhibiting substances on the mutation frequency of mice was investigated in vivo with the micronucleus test. The detergens Tween 80, vitamin A, and the antiphlogisticum phenylbutazone were used as DNA-repair inhibiting substances. Using the same irradiation doses, a statistic significant increase of mutagenicity respectively micronucleus frequency was found in high concentrations of Tween 80 and in all used dosages of vitamin A, but not in phenylbutazone and in low concentrations of tween. (auth.)

  6. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes...... such as extinct horses, cave bears, the marsupial wolf, the moa, and Neanderthal. In the past few years, this technology has been extended to the study of infectious disease in ancient Egyptian and South American mummies, the dietary habits of ancient animals, and agricultural practices and population dynamics......, and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived...

  7. Molecular dosimetry of chemical mutagens: measurement of molecular dose and DNA repair germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1975-01-01

    Molecular dosimetry in the germ cells of male mice is reviewed with regard to in vivo alkylation of sperm heads, in vivo alkylation of sperm DNA, and possible alkylation of sperm protamine. DNA repair in male germ cells is reviewed with regard to basic design of experiments, DNA repair in various stages of spermatogenesis, effect of protamine on DNA repair following treatment with EMS or x radiation, and induction of DNA repair by methyl methanesulfonate, propyl methanesulfonate, and isopropyl methanesulfonate

  8. 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

  9. DNA Mismatch Repair System: Repercussions in Cellular Homeostasis and Relationship with Aging

    Directory of Open Access Journals (Sweden)

    Juan Cristóbal Conde-Pérezprina

    2012-01-01

    Full Text Available The mechanisms that concern DNA repair have been studied in the last years due to their consequences in cellular homeostasis. The diverse and damaging stimuli that affect DNA integrity, such as changes in the genetic sequence and modifications in gene expression, can disrupt the steady state of the cell and have serious repercussions to pathways that regulate apoptosis, senescence, and cancer. These altered pathways not only modify cellular and organism longevity, but quality of life (“health-span”. The DNA mismatch repair system (MMR is highly conserved between species; its role is paramount in the preservation of DNA integrity, placing it as a necessary focal point in the study of pathways that prolong lifespan, aging, and disease. Here, we review different insights concerning the malfunction or absence of the DNA-MMR and its impact on cellular homeostasis. In particular, we will focus on DNA-MMR mechanisms regulated by known repair proteins MSH2, MSH6, PMS2, and MHL1, among others.

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

    International Nuclear Information System (INIS)

    Ammermann, D.

    1988-01-01

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

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  12. Genotoxic thresholds, DNA repair, and susceptibility in human populations

    International Nuclear Information System (INIS)

    Jenkins, Gareth J.S.; Zair, Zoulikha; Johnson, George E.; Doak, Shareen H.

    2010-01-01

    It has been long assumed that DNA damage is induced in a linear manner with respect to the dose of a direct acting genotoxin. Thus, it is implied that direct acting genotoxic agents induce DNA damage at even the lowest of concentrations and that no 'safe' dose range exists. The linear (non-threshold) paradigm has led to the one-hit model being developed. This 'one hit' scenario can be interpreted such that a single DNA damaging event in a cell has the capability to induce a single point mutation in that cell which could (if positioned in a key growth controlling gene) lead to increased proliferation, leading ultimately to the formation of a tumour. There are many groups (including our own) who, for a decade or more, have argued, that low dose exposures to direct acting genotoxins may be tolerated by cells through homeostatic mechanisms such as DNA repair. This argument stems from the existence of evolutionary adaptive mechanisms that allow organisms to adapt to low levels of exogenous sources of genotoxins. We have been particularly interested in the genotoxic effects of known mutagens at low dose exposures in human cells and have identified for the first time, in vitro genotoxic thresholds for several mutagenic alkylating agents (Doak et al., 2007). Our working hypothesis is that DNA repair is primarily responsible for these thresholded effects at low doses by removing low levels of DNA damage but becoming saturated at higher doses. We are currently assessing the roles of base excision repair (BER) and methylguanine-DNA methyltransferase (MGMT) for roles in the identified thresholds (Doak et al., 2008). This research area is currently important as it assesses whether 'safe' exposure levels to mutagenic chemicals can exist and allows risk assessment using appropriate safety factors to define such exposure levels. Given human variation, the mechanistic basis for genotoxic thresholds (e.g. DNA repair) has to be well defined in order that susceptible individuals are

  13. DNA repair studies in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.; Owens, J.G.

    1984-01-01

    In submammalian test systems, nitrosocarbamates (NEC) are 100-fold more mutagenic than are their corresponding nitrosourea homologues. To learn more about its interaction with germ-cell DNA in the mouse testis, male mice were given i.p. injections of NEC. Testicular injections of [ 3 H]dThd were given along with the NEC. Sixteen days after treatment, sperm were recovered from the caudal epididymides and assayed for an unscheduled-DNA-synthesis

  14. A comparison of the DNA and chromosome repair kinetics after #betta# irradiation

    International Nuclear Information System (INIS)

    Hittelman, W.N.; Pollard, M.

    1982-01-01

    The kinetics of repair at the chromosome and DNA levels were compared after #betta# irradiation of Chinese hamster ovary cells (CHO). Induction and repair of DNA damage were measured by the alkaline and neutral elution techniques, while chromosome damage and repair were determined by the technique of premature chromosome condensation. During and after #betta# irradiation, significant DNA repair occurred within 2 min. This fast repair could be inhibited by EDTA and pyrophosphate and probably reflected polynucleotide ligase activity. A slower component of DNA repair was detected between 15 and 60 min after irradiation, by which time most of the DNA had been repaired. In contrast, chromosome repair was not detectable until 45 min after irradiation, and nearly half of the chromatid breaks were repaired by 60 min. Cycloheximide, an inhibitor of protein synthesis, prevented chromosome break repair, yet had no effect on the immediate formation of chromatid exchanges or DNA repair. These results suggest the following: (1) the rapidly repairing DNA lesions are not important in the repair of chromosomes; (2) chromosome damage involves only a minority of the DNA lesions measured by alkaline and neutral DNA elution; and (3) chromosome repair may involve more than simply the repair of damaged DNA that can be detected by the alkaline and neutral elution assays

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

    Science.gov (United States)

    Gimenez, Estela; Manzano-Agugliaro, Francisco

    2017-10-30

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

  16. Coordinating repair of oxidative DNA damage with transcription and replication

    International Nuclear Information System (INIS)

    Cooper, P.K.

    2003-01-01

    Transcription-coupled repair (TCR) preferentially removes DNA lesions from template strands of active genes. Defects in TCR, which acts both on lesions removed by nucleotide excision repair (NER) and on oxidative lesions removed by base excision repair (BER), underlie the fatal developmental disorder Cockayne syndrome. Although its detailed mechanism remains unknown, TCR involves recognition of a stalled RNA polymerase (RNAP), removal or remodeling of RNAP to allow access to the lesion, and recruitment of repair enzymes. At a minimum, these early steps require a non-enzymatic function of the multifunctional repair protein XPG, the CSB protein with ATP-dependent chromatin remodeling activity, and the TFIIH complex (including the XPB and XPD helicases) that is also required for basal transcription initiation and NER. XPG exists in the cell in a complex with TFIIH, and in vitro evidence has suggested that it interacts with CSB. To address the mechanism of TCR, we are characterizing protein-DNA and protein-protein interactions of XPG. We show that XPG preferentially binds to double-stranded DNA containing bubbles resembling in size the unpaired regions associated with transcription. Two distinct domains of XPG are required for the observed strong binding specificity and stability. XPG both interacts directly with CSB and synergistically binds with it to bubble DNA, and it strongly stimulates the bubble DNA-dependent ATPase activity of CSB. Significantly for TCR, XPG also interacts directly with RNAP II, binds both the protein and nucleic acid components (the R-loop) of a stalled RNA polymerase, and forms a ternary complex with CSB and the stalled RNAP. These results are consistent with the model that XPG and CSB jointly interact with the DNA/chromatin structure in the vicinity of the stalled transcriptional apparatus and with the transcriptional machinery itself to remodel the chromatin and either move or remodel the blocked RNA polymerase to expose the lesion

  17. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

    Science.gov (United States)

    Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

  18. Involvement of the yeast DNA polymerase delta in DNA repair in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Giot, L. [State University of New York at Stony Brook, Stony Brook, NY. (United States); Chanet, R.; Simon, M.; Facca, C.; Faye, G.

    1997-08-15

    The POL3 encoded catalytic subunit of DNA polymerase delta possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24 degrees, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase delta. SDP5 is most probably the p55 subunit of Pol delta of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair. (author)

  19. Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage

    International Nuclear Information System (INIS)

    Park, Jung Wook; Nickel, Kwangok P.; Torres, Alexandra D.; Lee, Denis; Lambert, Paul F.; Kimple, Randall J.

    2014-01-01

    Background and purpose: Patients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV−) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV− HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown. Material and methods: Using immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot. Results: HPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation. Conclusions: Our findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV− HNC

  20. Distribution of DNA repair-related ESTs in sugarcane

    Directory of Open Access Journals (Sweden)

    W.C. Lima

    2001-12-01

    Full Text Available DNA repair pathways are necessary to maintain the proper genomic stability and ensure the survival of the organism, protecting it against the damaging effects of endogenous and exogenous agents. In this work, we made an analysis of the expression patterns of DNA repair-related genes in sugarcane, by determining the EST (expressed sequence tags distribution in the different cDNA libraries of the SUCEST transcriptome project. Three different pathways - photoreactivation, base excision repair and nucleotide excision repair - were investigated by employing known DNA repair proteins as probes to identify homologous ESTs in sugarcane, by means of computer similarity search. The results showed that DNA repair genes may have differential expressions in tissues, depending on the pathway studied. These in silico data provide important clues on the potential variation of gene expression, to be confirmed by direct biochemical analysis.As vias de reparo de DNA são requeridas para manter a necessária estabilidade genômica e garantir a sobrevivência do organismo, frente aos efeitos deletérios causados por fatores endógenos e exógenos. Neste trabalho, realizamos a análise dos padrões de expressão dos genes de reparo de DNA encontrados na cana-de-açúcar, pela determinação da distribuição de ESTs nas diferentes bibliotecas de cDNA no projeto de transcriptoma SUCEST. Três vias de reparo - fotorreativação, reparo por excisão de bases e reparo por excisão de nucleotídeos - foram estudadas através do uso de proteínas de reparo como sondas para identificação de ESTs homólogos em cana-de-açúcar, com base na procura computacional de similaridade. Os resultados indicam que os genes de reparo de DNA possuem uma expressão diferencial nos tecidos, dependendo da via de reparo analisada. Esses dados in silico fornecem importantes indícios da expressão diferencial, a qual deve ser confirmada por análises bioquímicas diretas.

  1. Chemotherapeutic Drugs: DNA Damage and Repair in Glioblastoma.

    Science.gov (United States)

    Annovazzi, Laura; Mellai, Marta; Schiffer, Davide

    2017-05-26

    Despite improvements in therapeutic strategies, glioblastoma (GB) remains one of the most lethal cancers. The presence of the blood-brain barrier, the infiltrative nature of the tumor and several resistance mechanisms account for the failure of current treatments. Distinct DNA repair pathways can neutralize the cytotoxicity of chemo- and radio-therapeutic agents, driving resistance and tumor relapse. It seems that a subpopulation of stem-like cells, indicated as glioma stem cells (GSCs), is responsible for tumor initiation, maintenance and recurrence and they appear to be more resistant owing to their enhanced DNA repair capacity. Recently, attention has been focused on the pivotal role of the DNA damage response (DDR) in tumorigenesis and in the modulation of therapeutic treatment effects. In this review, we try to summarize the knowledge concerning the main molecular mechanisms involved in the removal of genotoxic lesions caused by alkylating agents, emphasizing the role of GSCs. Beside their increased DNA repair capacity in comparison with non-stem tumor cells, GSCs show a constitutive checkpoint expression that enables them to survive to treatments in a quiescent, non-proliferative state. The targeted inhibition of checkpoint/repair factors of DDR can contribute to eradicate the GSC population and can have a great potential therapeutic impact aiming at sensitizing malignant gliomas to treatments, improving the overall survival of patients.

  2. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations

    NARCIS (Netherlands)

    Sijmons, Rolf H.; Hofstra, Robert M. W.

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive

  3. Polymorphisms in human DNA repair genes and head and neck ...

    Indian Academy of Sciences (India)

    Abstract. Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and ... Such studies may benefit from analysis of multiple genes or polymorphisms and from the ... low survival and high morbidity when diagnosed in advanced ...... racial and/or ethnic cohort.

  4. Biomarkers of oxidative damage to DNA and repair

    DEFF Research Database (Denmark)

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

    2008-01-01

    environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer...

  5. Principles of ubiquitin and SUMO modifications in DNA repair

    NARCIS (Netherlands)

    Bergink, Steven; Jentsch, Stefan

    2009-01-01

    With the discovery in the late 1980s that the DNA-repair gene RAD6 encodes a ubiquitin-conjugating enzyme, it became clear that protein modification by ubiquitin conjugation has a much broader significance than had previously been assumed. Now, two decades later, ubiquitin and its cousin SUMO are

  6. p53 downregulates the Fanconi anaemia DNA repair pathway.

    Science.gov (United States)

    Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

    2016-04-01

    Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

  7. DNA repair and the genetic control of radiosensitivity in yeast

    International Nuclear Information System (INIS)

    Haynes, R.H.

    1975-01-01

    The following topics are discussed: advantages of yeasts for easily manipulated model systems for studies on molecular biology of eukaryotes; induction of x-ray-resistant mutants by radiations and chemicals; genetics of uv-sensitive mutants; loci of genes affecting radiosensitivity; gene interactions in multiple mutants; liquid-holding recovery; mitotic and meiotic recombination; and repair of yeast mitochondrial DNA

  8. Altered DNA repair, oxidative stress and antioxidant status

    Indian Academy of Sciences (India)

    Coronary artery disease (CAD) is a multifactorial disease caused by the interplay of environmental risk factors with multiple predisposing genes. The present study was undertaken to evaluate the role of DNA repair efficiency and oxidative stress and antioxidant status in CAD patients. Malonaldehyde (MDA), which is an ...

  9. Defective DNA repair mechanisms in prostate cancer: impact of olaparib

    Directory of Open Access Journals (Sweden)

    De Felice F

    2017-03-01

    Full Text Available Francesca De Felice,1 Vincenzo Tombolini,1 Francesco Marampon,2 Angela Musella,3 Claudia Marchetti3 1Department of Radiotherapy, Policlinico Umberto I, “Sapienza” University of Rome, Rome, 2Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L’Aquila, L’Aquila, 3Department of Gynecological and Obstetrical Sciences and Urological Sciences, “Sapienza” University of Rome, Rome, Italy Abstract: The field of prostate oncology has continued to change dramatically. It has truly become a field that is intensely linked to molecular genetic alterations, especially DNA-repair defects. Germline breast cancer 1 gene (BRCA1 and breast cancer 2 gene (BRCA2 mutations are implicated in the highest risk of prostate cancer (PC predisposition and aggressiveness. Poly adenosine diphosphate ribose polymerase (PARP proteins play a key role in DNA repair mechanisms and represent a valid target for new therapies. Olaparib is an oral PARP inhibitor that blocks DNA repair pathway and coupled with BRCA mutated-disease results in tumor cell death. In phase II clinical trials, including patients with advanced castration-resistant PC, olaparib seems to be efficacious and well tolerated. Waiting for randomized phase III trials, olaparib should be considered as a promising treatment option for PC. Keywords: prostate cancer, metastatic disease, castration resistant, BRCA, DNA-repair, PARP, olaparib

  10. Xeroderma pigmentosum: recent studies on the DNA repair defects

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1978-01-01

    Xeroderma pigmentosum is a recessive autosomal disease of humans that is characterized by a high prevalence of skin cancers. Results of studies on cells from such patients indicate a defect in the repair of DNA damage associated with exposure to ultraviolet radiation. Since this observation was reported, a large amount of information on this disease has accumulated in the literature

  11. Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic endonuclease 1

    DEFF Research Database (Denmark)

    Yang, Jenq-Lin; Tadokoro, Takashi; Keijzers, Guido

    2010-01-01

    inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species...

  12. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    Science.gov (United States)

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  14. High LET radiation and mechanism of DNA damage repair

    International Nuclear Information System (INIS)

    Furusawa, Yoshiya

    2004-01-01

    Clarifying the mechanism of repair from radiation damage gives most important information on radiation effects on cells. Approximately 10% of biological experiments groups in Heavy Ion Medical Accelerator in Chiba (HIMAC) cooperative research group has performed the subject. They gave a lot of new findings on the mechanism, and solved some open questions. The reason to show the peak of relative biological effectiveness RBE at around 100-200 keV/μm causes miss-repair of DNA damage. Sub-lethal damage generated by high linear energy transfer (LET) radiation can be repaired fully. Potentially lethal damages by high-LET radiation also repaired, but the efficiency decreased with the LET, and so on. (author)

  15. 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.

  16. 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.

  17. DNA repair in spermatocytes and spermatids of the mouse

    International Nuclear Information System (INIS)

    Sega, G.A.

    1982-01-01

    When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced

  18. DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

    Science.gov (United States)

    Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

    2012-03-01

    Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends.

    Science.gov (United States)

    Das, Ushati; Chauleau, Mathieu; Ordonez, Heather; Shuman, Stewart

    2014-08-05

    Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

  20. The Bright and the Dark Sides of DNA Repair in Stem Cells

    OpenAIRE

    Frosina, Guido

    2010-01-01

    DNA repair is a double-edged sword in stem cells. It protects normal stem cells in both embryonic and adult tissues from genetic damage, thus allowing perpetuation of intact genomes into new tissues. Fast and efficient DNA repair mechanisms have evolved in normal stem and progenitor cells. Upon differentiation, a certain degree of somatic mutations becomes more acceptable and, consequently, DNA repair dims. DNA repair turns into a problem when stem cells transform and become cancerous. Tran...

  1. Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

    Science.gov (United States)

    Jobert, Laure; Nilsen, Hilde

    2014-07-01

    The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

  2. Reduction in DNA repair capacity following differentiation of murine proadipocytes

    International Nuclear Information System (INIS)

    Tofilon, P.J.; Meyn, R.E.

    1988-01-01

    It has been suggested that terminally differentiated mammalian cells have a decreased DNA repair capacity, compared with proliferating stem cells. To investigate this hypothesis, we have examined γ-ray-induced DNA strand breaks and their repair in the murine proadipocyte stem cell line 3T3-T. By exposure to human plasma, 3T3-T cells can be induced to undergo nonterminal and then terminal differentiation. DNA strand breaks were evaluated using the technique of alkaline elution. No difference was detected among stem, nonterminally differentiated, and terminally differentiated cells in the initial levels of radiation-induced DNA strand breaks. Each of the strand break dose responses increased as a linear function of γ-ray dose. The strand breaks induced by 4 Gy rejoined following biphasic kinetics for each cell type. At each time point examined after irradiation, however, the percentage of strand breaks that had not rejoined in terminally differentiated cells was three to six times greater than in stem cells. The rate of strand break rejoining in nonterminally differentiated cells was of an intermediate value between that of the stem and of the terminally differentiated cells. These results indicate that, at least for 3T3-T cells, differentiated cells have a reduced capacity for DNA repair

  3. Interplay of DNA repair with transcription: from structures to mechanisms.

    Science.gov (United States)

    Deaconescu, Alexandra M; Artsimovitch, Irina; Grigorieff, Nikolaus

    2012-12-01

    Many DNA transactions are crucial for maintaining genomic integrity and faithful transfer of genetic information but remain poorly understood. An example is the interplay between nucleotide excision repair (NER) and transcription, also known as transcription-coupled DNA repair (TCR). Discovered decades ago, the mechanisms for TCR have remained elusive, not in small part due to the scarcity of structural studies of key players. Here we summarize recent structural information on NER/TCR factors, focusing on bacterial systems, and integrate it with existing genetic, biochemical, and biophysical data to delineate the mechanisms at play. We also review emerging, alternative modalities for recruitment of NER proteins to DNA lesions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Base excision repair in Archaea: back to the future in DNA repair.

    Science.gov (United States)

    Grasso, Stefano; Tell, Gianluca

    2014-09-01

    Together with Bacteria and Eukarya, Archaea represents one of the three domain of life. In contrast with the morphological difference existing between Archaea and Eukarya, these two domains are closely related. Phylogenetic analyses confirm this evolutionary relationship showing that most of the proteins involved in DNA transcription and replication are highly conserved. On the contrary, information is scanty about DNA repair pathways and their mechanisms. In the present review the most important proteins involved in base excision repair, namely glycosylases, AP lyases, AP endonucleases, polymerases, sliding clamps, flap endonucleases, and ligases, will be discussed and compared with bacterial and eukaryotic ones. Finally, possible applications and future perspectives derived from studies on Archaea and their repair pathways, will be taken into account. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Repair of human DNA: radiation and chemical damage in normal and xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Regan, J.D.; Setlow, R.B.

    1976-01-01

    We present the experimental evidence we have gathered, using a particular assay for DNA repair in human cells, the photolysis of bromodeoxyuridine (BrdUrd) incorporated during repair. This assay characterizes the sequence of repair events that occur in human cells after radiation, both ultraviolet and ionizing, and permits an estimation of the size of the average repaired region after these physical insults to DNA. We will discuss chemical insults to DNA and attempt to liken the repair processes after chemical damages of various kinds to those repair processes that occur in human DNA after damage from physical agents. We will also show results indicating that, under certain conditions, repair events resembling those seen after uv-irradiation can be observed in normal human cells after ionizing radiation. Furthermore the XP cells, defective in the repair of uv-induced DNA damage, show defective repair of these uv-like DNA lesions induced by ionizing radiation

  6. Activation of DNA damage repair pathways by murine polyomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Katie; Nicholas, Catherine; Garcea, Robert L., E-mail: Robert.Garcea@Colorado.edu

    2016-10-15

    Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.

  7. DNA replication and repair of Tilapia cells: Pt. 2

    International Nuclear Information System (INIS)

    Chen, J.D.; Yew, F.H.

    1988-01-01

    TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34 0 C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 0 C to the sublethal high temperature of 37 0 C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37 0 C the rate of DNA synthesis overshot the 31 0 C control level by 180%. When moved to low temperature (18 0 C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25 0 C, but not at 18 0 C. Initiation of nascent DNA synthesis was blocked at 4Jm -2 in TO-2 cells compared with ≤ 1Jm -2 in mammalian cells. After 9Jm -2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair. (author)

  8. Measurement of DNA repair deficiency in workers exposed to benzene

    International Nuclear Information System (INIS)

    Hallberg, L.M.; Au, W.W.; El Zein, R.; Grossman, L.

    1996-01-01

    We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m 2 UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m 2 and 350 J/m 2 were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (<0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. 26 refs., 4 figs., 2 tabs

  9. Transfer of Chinese hamster DNA repair gene(s) into repair-deficient human cells (Xeroderma pigmentosum)

    International Nuclear Information System (INIS)

    Karentz, D.; Cleaver, J.E.

    1985-01-01

    Transfer of repair genes by DNA transfection into repair-deficient Xeroderma pigmentosum (XP) cells has thus far been unsuccessful, presenting an obstacle to cloning XP genes. The authors chose an indirect route to transfer repair genes in chromosome fragments. DNA repair-competent (UV resistant) hybrid cell lines were established by PEG-mediated fusions of DNA repair-deficient (UV sensitive) human fibroblasts (XP12RO) with wild type Chinese hamster (CHO) cells (AA8). CHO cells were exposed to 5 Krad X-rays prior to fusions, predisposing hybrid cells to lose CHO chromosome fragments preferentially. Repair-competent hybrids were selected by periodic exposures to UV light. Secondary and tertiary hybrid cell lines were developed by fusion of X-irradiated hybrids to XP12RO. The hybrid cell lines exhibit resistance to UV that is comparable to that of CHO cells and they are proficient at repair replication after UV exposure. Whole cell DNA-DNA hybridizations indicate that the hybrids have greater homology to CHO DNA than is evident between XP12RO and CHO. These observations indicate that CHO DNA sequences which can function in repair of UV-damaged DNA in human cells have been transferred into the genome of the repair-deficient XP12RO cells

  10. The repair of damage to DNA in different cell types

    International Nuclear Information System (INIS)

    Karran, P.

    1974-01-01

    DNA single strand breaks induced by either X-ray irradiation or by methyl methanesulphonate (MMS) were studied in different lymphoid cell populations directly taken from the animal and maintained in tissue culture merely for the duration of the experiment. The results obtained from these cell populations were compared with those obtained with L5178Y cells maintained in tissue culture. All cell types studied were found to possess at least one class of enzymes required for repair of DNA damage, namely those enzymes involved in the rejoining of X-ray induced by MMS is different in each cell type. Repair replication was at much reduced levels and the endonucleolytic degradation was at much reduced levels and the endonucleolytic degradation was initiated at lower MMS concentration in the lymphoid cells as compared to L5178Y cells. It is suggested that the overall ''repair capacity'' of a population may be related to the number of cells in a cycle which, moreover, might be the only ones to have the ability to repair damage to DNA induced by MMS (G.G.)

  11. DNA-radiosensitivity and repair in mammolian cells

    International Nuclear Information System (INIS)

    Proskuryakov, S.Ya.; Ivannik, B.P.; Ryabchenko, N.I.

    1979-01-01

    Determination was made of the formation and repair of single-stranded DNA breaks (SB) in cells of rat thymus and liver and Ehrlich's ascites tumor (EAT) with the use of the method of low-gradient viscosimetry of alkaline cell lysates. The radiochemical yield of single-stranded breaks (Gsub(SB)) induced by irradiation of animals is 41.2 eV/break for hepatocytes, 96.8 eV/break, for thymocytes, and 129.7 eV/break, for EAT cells. The half-recovery time of single-stranded DNA breaks for cells of thymus and EAT exposed in vivo is 16.0 and 5.1 s -1 , correspondingly. In hepatocytes exposed in vivo and in vitro no repairs occurs for 3 h. Under conditions of inhibition of SB repair, when suspensions of thymocytes and hepatocytes were exposed in vitro at 4 deg C, Gsub(SB) is 35.5 and 38.7 eV/break, respectively. The analysis of the data obtained prompts the conclusion that under in vivo conditions, there is a correlation between DNA radiosensitivity and the rate of repair processes

  12. 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.

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

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

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

  14. Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA

    DEFF Research Database (Denmark)

    Skarpengland, Tonje; B. Dahl, Tuva; Skjelland, Mona

    2016-01-01

    Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER...

  15. Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12

    International Nuclear Information System (INIS)

    Strike, P.; Roberts, R.J.

    1982-01-01

    The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA + and uvrB + gene products, but not the host recA + gene product. The requirement for both homologous DNA and the uvrA + gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered

  16. DNA repair deficiency in lymphocytes from patients with actinic keratosis

    International Nuclear Information System (INIS)

    Abo-Darub, J.M.; Mackie, R.; Pitts, J.D.

    1978-01-01

    DNA repair activity was measured in peripheral blood lymphocytes from 18 patients with Actinic Keratosis and 18 age-matched control subjects, by comparing the incorporation of 3 H-thymidine into cells after irradiation with ultraviolet light with that into unirradiated cells. The incorporation was followed autoradiographically or by measuring acid insoluble radioactivity in cells labelled in the presence of hydroxyurea. The repair activity in lymphocytes from Actinic keratosis patients was only 47.1% (+-6.5%) of that in cells from the control subjects

  17. Impact of radiotherapy on PBMCs DNA repair capacity - Use of a multiplexed functional repair assay

    International Nuclear Information System (INIS)

    Sauvaigo, S.; Sarrazy, F.; Breton, J.; Caillat, S.; Chapuis, V.

    2012-01-01

    Radiation therapy is an essential part of cancer treatment as about 50% of patients will receive radiations at least once. Significant broad variation in radiosensitivity has been demonstrated in patients. About 5-10% of patients develop acute toxicity after radiotherapy. Therefore there is a need for the identification of markers able to predict the occurrence of adverse effects and thus adapt the radiotherapy regimen for radiosensitive patients. As a first step toward this goal, and considering the DNA repair defects associated with hypersensitivity radiation syndromes, we investigated the DNA repair phenotype of patients receiving radiotherapy. More precisely, we used a functional repair assay on support to follow the evolution of the glycosylases/AP endonuclease activities of PBMCs extracts of a series of patients during the time course of radiotherapy. For each patient, we collected one PBMCs sample before the first radiotherapy application (S1) and three samples after (S2 to S4) (one day and one week after application 1, and one at the end of the radiotherapy protocol). These four samples have been analysed for 11 donors. Clustering analyses of the results demonstrated a great heterogeneity of responses among the patients. Interestingly, this heterogeneity decreased between S1 and S4 where only 2 classes of patients remained if we except one patient that exhibited an atypical DNA repair phenotype. Furthermore, we showed that repair of several oxidized bases significantly increased between S1 and S3 or S4 (8oxoG, thymine glycol, A paired with 8oxoG), suggesting an adaptation of patients repair systems to the oxidative stress generated by the ionising radiations. Our preliminary results provided evidence that the DNA repair phenotype was impacted by the radiotherapy regimen. Further characterization of patients with known repair defects are needed to determine if atypical repair phenotypes could be associated with radiotherapy complications. Finally

  18. Emerging critical roles of Fe-S clusters in DNA replication and repair

    Science.gov (United States)

    Fuss, Jill O.; Tsai, Chi-Lin; Ishida, Justin P.; Tainer, John A.

    2015-01-01

    Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life – the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. PMID:25655665

  19. Aspects of DNA repair and nucleotide pool imbalance

    Energy Technology Data Exchange (ETDEWEB)

    Holliday, R.

    1985-01-01

    Evidence that optimum repair depends on adequate pools of deoxynucleotide triphosphates (dNTPs) comes from the study of pyrimidine auxotrophs of Ustilago maydis. These strains are sensitive to UV light and X-rays, and for pyr1-1 it has been shown that the intracellular concentration of dTTP is reduced about 7-fold. The survival curve of pyr1-1 after UV-treatment, and split dose experiments with wild-type cells, provide evidence for an inducible repair mechanism, which probably depends on genetic recombination. Although inducible repair saves cellular resources, it has the disadvantage of becoming ineffective at doses which are high enough to inactivate the repressed structural gene(s) for repair enzymes. It is clear that a wide variety of repair mechanisms have evolved to remove lesions which arise either spontaneously or as a result of damage from external agents. Nevertheless, it would be incorrect to assume that all species require all possible pathways of repair. It is now well established that the accuracy of DNA and protein synthesis depends on proof-reading or editing mechanisms. Optimum accuracy levels will evolve from the balance between error avoidance in macromolecular synthesis and physiological efficiency in growth and propagation.

  20. Human diseases with genetically altered DNA repair processes

    International Nuclear Information System (INIS)

    Cleaver, J.E.; Bootsma, D.; Friedberg, E.

    1975-01-01

    DNA repair of single-strand breaks (produced by ionizing radiation) and of base damage (produced by ultraviolet (uv) light) are two repair mechanisms that most mammalian cells possess. Genetic defects in these repair mechanisms are exemplified by cells from the human premature-aging disease, progeria, which fail to rejoin single-strand breaks, and the skin disease, xeroderma pigmentosum (XP), which exhibits high actinic carcinogenesis and involves failure to repair base damage. In terms of the response of XP cells, many chemical carcinogens can be classified as either x-ray-like (i.e., they cause damage that XP cells can repair) or uv-like (i.e., they cause damage that XP cells cannot repair). The first group contains some of the more strongly carcinogenic chemicals (e.g., alkylating agents). XP occurs in at least two clinical forms, and somatic cell hybridization indicates at least three complementation groups. In order to identify cell lines from various different laboratories unambiguously, a modified nomenclature of XP lines is proposed. (U.S.)

  1. Human diseases with genetically altered DNA repair processes

    International Nuclear Information System (INIS)

    Cleaver, J.E.; Bootsma, D.; Friedberg, E.

    1975-01-01

    DNA repair of single-strand breaks (produced by ionizing radiation) and of base damage (produced by ultraviolet (UV) light) are two repair mechanisms that most mammalian cells possess. Genetic defects in these repair mechanisms are exemplified by cells from the human premature-aging disease, progeria, which fail to rejoin single-strand breaks, and the skin disease, xeroderma pigmentosum (XP), which exhibits high actinic carcinogenesis and involves failure to repair base damage. In terms of the response of XP cells, many chemical carcinogens can be classified as either X-ray-like (i.e., they cause damage that XP cells can repair) or UV-like (i.e., they cause damage that XP cells cannot repair). The first group contains some of the more strongly carcinogenic chemicals (e.g., alkylating agents). XP occurs in at least two clinical forms, and somatic cell hybridization indicates at least three complementation groups. In order to identify cell lines from various different laboratories unambiguously, a modified nomenclature of XP lines is proposed

  2. Kinetics and mechanism of DNA repair; Evaluation of caged compounds for use in studies of u. v. -induced DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Meldrum, R.A.; Wharton, C.W. (Birmingham Univ. (UK). Dept. of Biochemistry); Shall, S. (Sussex Univ., Brighton (UK). School of Biological Sciences)

    1990-03-15

    Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the {gamma}-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside trisphosphate is isotopically labelled in the {alpha}-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair. (author).

  3. Role of DNA lesions and DNA repair in mutagenesis by carcinogens in diploid human fibroblasts

    International Nuclear Information System (INIS)

    Maher, V.M.; McCormick, J.J.

    1986-01-01

    The authors investigated the cytotoxicity, mutagenicity, and transforming activity of carcinogens and radiation in diploid human fibroblasts, using cells which differ in their DNA repair capacity. The results indicate that cell killing and induction of mutations are correlated with the number of specific lesions remaining unrepaired in the cells at a particular time posttreatment. DNA excision repair acts to eliminate potentially cytotoxic and mutagenic (and transforming) damage from DNA before these can be converted into permanent cellular effects. Normal human fibroblasts were derived from skin biopsies or circumcision material. Skin fibroblasts from xeroderma pigmentosum (XP) patients provided cells deficient in nucleotide excision repair of pyrimidine dimers or DNA adducts formed by bulky ring structures. Cytotoxicity was determined from loss of ability to form a colony. The genetic marker used was resistance to 6-thioguanine (TG). Transformation was measured by determining the frequency of anchorage-independent cells

  4. Radiosensitivity, radio-curability and DNA repair

    International Nuclear Information System (INIS)

    Vogin, G.

    2011-01-01

    Improvements in accuracy stand as the heart of the success of today's radiotherapy. The dose may be delivered with a sub millimetric accuracy, may also conform to complex shapes, or track external and internal organ motions. In parallel, we may increase the tumour's radio-curability by modulating the biological effects generated by ionizing radiation into the patient. It was precisely the topic of the 2009 Lucien-Mallet prize organized by the French Society for Radiation Oncology (SFRO) and the Centre Antoine-Beclere under the auspices of the Fondation de France. In this review we will precisely describe the integrated molecular response to ionizing radiations. Starting from early observations, we are going to introduce the concept of cellular radiosensitivity as the global response of the irradiated cell. We will then focus into the cell and especially its nucleus. We will describe here the most complex and deleterious radioinduced damages. In the next chapter, we will dissect the molecular pathway that aims to detect and repair the previous lesions. The last part of the review will finally deal with the diagnostic, prognostic and therapeutic impacts emerging from the alliance between clinical and molecular radiobiology. (author)

  5. 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)

  6. Repair of oxidative DNA damage by amino acids.

    Science.gov (United States)

    Milligan, J R; Aguilera, J A; Ly, A; Tran, N Q; Hoang, O; Ward, J F

    2003-11-01

    Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

  7. Targeting telomerase and DNA repair in human cancers

    International Nuclear Information System (INIS)

    Prakash Hande, M.

    2014-01-01

    Telomerase reactivation is essential for telomere maintenance in human cancer cells ensuring indefinite proliferation. Targeting telomere homeostasis has become one of the promising strategies in the therapeutic management of tumours. One major potential drawback, however, is the time lag between telomerase inhibition and critically shortened telomeres triggering cell death, allowing cancer cells to acquire drug resistance. Numerous studies over the last decade have highlighted the role of DNA repair proteins such as Poly (ADP-Ribose) Polymerase-1 (PARP-1), and DNA-dependent protein kinase (DNA-PKcs) in the maintenance of telomere homoeostasis. Dysfunctional telomeres, resulting from the loss of telomeric DNA repeats or the loss of function of telomere-associated proteins trigger DNA damage responses similar to that observed for double strand breaks. We have been working on unravelling such synthetic lethality in cancer cells and this talk would be on one such recently concluded study that demonstrates that inhibition of DNA repair pathways, i.e., NHEJ pathway and that of telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. (author)

  8. DNA repair in neurons: So if they don't divide what's to repair?

    Energy Technology Data Exchange (ETDEWEB)

    Fishel, Melissa L. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States) and Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States) and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States)]. E-mail: mkelley@iupui.edu

    2007-01-03

    Neuronal DNA repair remains one of the most exciting areas for investigation, particularly as a means to compare the DNA repair response in mitotic (cancer) vs. post-mitotic (neuronal) cells. In addition, the role of DNA repair in neuronal cell survival and response to aging and environmental insults is of particular interest. DNA damage caused by reactive oxygen species (ROS) such as generated by mitochondrial respiration includes altered bases, abasic sites, and single- and double-strand breaks which can be prevented by the DNA base excision repair (BER) pathway. Oxidative stress accumulates in the DNA of the human brain over time especially in the mitochondrial DNA (mtDNA) and is proposed to play a critical role in aging and in the pathogenesis of several neurological disorders including Parkinson's disease, ALS, and Alzheimer's diseases. Because DNA damage accumulates in the mtDNA more than nuclear DNA, there is increased interest in DNA repair pathways and the consequence of DNA damage in the mitochondria of neurons. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the BER pathway. Following the notion that the bulk of neuronal DNA damage is acquired by oxidative DNA damage and ROS, the BER pathway is a likely area of focus for neuronal studies of DNA repair. BER variations in brain aging and pathology in various brain regions and tissues are presented. Therefore, the BER pathway is discussed in greater detail in this review than other repair pathways. Other repair pathways including direct reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination and non-homologous end joining are also discussed. Finally, there is a growing interest in the role that DNA repair pathways play in the clinical arena as they relate to the neurotoxicity and neuropathy associated with cancer treatments. Among the numerous side effects of cancer treatments, major

  9. N-Butyrate alters chromatin accessibility to DNA repair enzymes

    International Nuclear Information System (INIS)

    Smith, P.J.

    1986-01-01

    Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells

  10. Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

    Science.gov (United States)

    Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

    2016-01-01

    Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

  11. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  12. The influence of radio- and chemotherapy on DNA repair of peripheral lymphocytes of tumor patients

    International Nuclear Information System (INIS)

    Klein, W.; Alth, G.; Klein, H.; Koren, H.

    1979-07-01

    The influence of radiotherapy and chemotherapy, respectively, on DNA excision repair was investigated in lymphocytes of the peripheral blood of 10 and 5 patients with malignancies. No effects on DNA repair were found using only betatrone of 60 Co-irradiation under normal conditions. Combination of both irradiation schedules over a longer period of therapy provoked an inhibition of DNA repair. Chemotherapy inhibits DNA repair immediately after starting therapy, but after relatively short time, the extent of DNA repair increases above normal level. (author)

  13. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-02-21

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-12-01

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

  16. Involvement of DNA mismatch repair in the maintenance of heterochromatic DNA stability in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Basanta K Dahal

    2017-10-01

    Full Text Available Heterochromatin contains a significant part of nuclear DNA. Little is known about the mechanisms that govern heterochromatic DNA stability. We show here that in the yeast Saccharomyces cerevisiae (i DNA mismatch repair (MMR is required for the maintenance of heterochromatic DNA stability, (ii MutLα (Mlh1-Pms1 heterodimer, MutSα (Msh2-Msh6 heterodimer, MutSβ (Msh2-Msh3 heterodimer, and Exo1 are involved in MMR at heterochromatin, (iii Exo1-independent MMR at heterochromatin frequently leads to the formation of Pol ζ-dependent mutations, (iv MMR cooperates with the proofreading activity of Pol ε and the histone acetyltransferase Rtt109 in the maintenance of heterochromatic DNA stability, (v repair of base-base mismatches at heterochromatin is less efficient than repair of base-base mismatches at euchromatin, and (vi the efficiency of repair of 1-nt insertion/deletion loops at heterochromatin is similar to the efficiency of repair of 1-nt insertion/deletion loops at euchromatin.

  17. 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

  18. 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.

  19. DNA repair, human cancer and assessment of radiation hazards

    International Nuclear Information System (INIS)

    Paterson, M.C.; Myers, D.K.

    1979-09-01

    Cancers, like genetic defects, are thought to be caused primarily by changes in DNA. Part of the evidence in support of this hypothesis derives from the study of certain rare hereditary disorders in man associated with high risk of cancer. Cells derived from patients suffering from at least one of these disorders, ataxia telangiectasia, appear to be defective in their ability to repair the damage caused by radiation and/or certain other environmental agents. Studies of the consequences of DNA repair suggest that currently accepted estimates of the carcinogenic hazards of low level radiation are substantially correct. There would appear to be some margin of safety involved in these risk estimates for the majority of the population, but any major reduction in the currently accepted risk estimates appears inadvisable in view of the existence of potentially radiosensitive subgroups forming a minority in the general population. (author)

  20. Inducible DNA-repair systems in yeast: competition for lesions.

    Science.gov (United States)

    Mitchel, R E; Morrison, D P

    1987-03-01

    DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate

  1. True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

    Directory of Open Access Journals (Sweden)

    Nicolas Le May

    2010-01-01

    Full Text Available Nucleotide excision repair (NER is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

  2. Dissecting DNA repair in adult high grade gliomas for patient stratification in the post-genomic era

    Science.gov (United States)

    Perry, Christina; Agarwal, Devika; Abdel-Fatah, Tarek M.A.; Lourdusamy, Anbarasu; Grundy, Richard; Auer, Dorothee T.; Walker, David; Lakhani, Ravi; Scott, Ian S.; Chan, Stephen; Ball, Graham; Madhusudan, Srinivasan

    2014-01-01

    Deregulation of multiple DNA repair pathways may contribute to aggressive biology and therapy resistance in gliomas. We evaluated transcript levels of 157 genes involved in DNA repair in an adult glioblastoma Test set (n=191) and validated in ‘The Cancer Genome Atlas’ (TCGA) cohort (n=508). A DNA repair prognostic index model was generated. Artificial neural network analysis (ANN) was conducted to investigate global gene interactions. Protein expression by immunohistochemistry was conducted in 61 tumours. A fourteen DNA repair gene expression panel was associated with poor survival in Test and TCGA cohorts. A Cox multivariate model revealed APE1, NBN, PMS2, MGMT and PTEN as independently associated with poor prognosis. A DNA repair prognostic index incorporating APE1, NBN, PMS2, MGMT and PTEN stratified patients in to three prognostic sub-groups with worsening survival. APE1, NBN, PMS2, MGMT and PTEN also have predictive significance in patients who received chemotherapy and/or radiotherapy. ANN analysis of APE1, NBN, PMS2, MGMT and PTEN revealed interactions with genes involved in transcription, hypoxia and metabolic regulation. At the protein level, low APE1 and low PTEN remain associated with poor prognosis. In conclusion, multiple DNA repair pathways operate to influence biology and clinical outcomes in adult high grade gliomas. PMID:25026297

  3. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  4. Metabolism, genomics, and DNA repair in the mouse aging liver

    DEFF Research Database (Denmark)

    Lebel, Michel; de Souza-Pinto, Nadja C; Bohr, Vilhelm A

    2011-01-01

    hepatic metabolic and detoxification activities, with implications for systemic aging and age-related disease. It has become clear, using rodent models as biological tools, that genetic instability in the form of gross DNA rearrangements or point mutations accumulate in the liver with age. DNA lesions......The liver plays a pivotal role in the metabolism of nutrients, drugs, hormones, and metabolic waste products, thereby maintaining body homeostasis. The liver undergoes substantial changes in structure and function within old age. Such changes are associated with significant impairment of many......, such as oxidized bases or persistent breaks, increase with age and correlate well with the presence of senescent hepatocytes. The level of DNA damage and/or mutation can be affected by changes in carcinogen activation, decreased ability to repair DNA, or a combination of these factors. This paper covers some...

  5. Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects.

    Science.gov (United States)

    Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

    2008-12-15

    Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines

  6. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

    International Nuclear Information System (INIS)

    Dreslor, S.L.; Frattini, M.G.

    1987-01-01

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

  7. DNA damage and nucleotide excision repair capacity in healthy individuals

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Naccarati, Alessio; Poláková, Veronika; Pardini, Barbara; Vodičková, Ludmila; Štětina, R.; Schmuczerová, Jana; Šmerhovský, Z.; Lipská, L.; Vodička, Pavel

    2011-01-01

    Roč. 25, č. 7 (2011), s. 511-517 ISSN 0893-6692 R&D Projects: GA ČR GAP304/10/1286; GA MŠk 7F10069 Grant - others:GA MŠk(CZ) GAUK124710 Institutional research plan: CEZ:AV0Z50390512 Keywords : BPDE-induced DNA repair capacity * comet assay * interindividual variability Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.709, year: 2011

  8. Clinical Radiation Sensitivity With DNA Repair Disorders: An Overview

    International Nuclear Information System (INIS)

    Pollard, Julianne M.; Gatti, Richard A.

    2009-01-01

    Adverse reactions to radiotherapy represent a confounding phenomenon in radiation oncology. These reactions are rare, and many have been associated with individuals with DNA repair disorders such as ataxia-telangiectasia and Nijmegen Breakage syndrome. A paucity of published data is available detailing such circumstances. This overview describes four exemplary situations, a comprehensive list of 32 additional cases, and some insights gleaned from this overall experience. Fanconi anemia was associated with more than one-half of the reports. The lowest dose given to a patient that resulted in a reaction was 3 Gy, given to an ataxia-telangiectasia patient. Most patients died within months of exposure. It is clear that the patients discussed in this report had complicated illnesses, in addition to cancer, and the radiotherapy administered was most likely their best option. However, the underlying DNA repair defects make conventional radiation doses dangerous. Our findings support previous wisdom that radiotherapy should either be avoided or the doses should be selected with great care in the case of these radiosensitive genotypes, which must be recognized by their characteristic phenotypes, until more rapid, reliable, and functional assays of DNA repair become available.

  9. DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

    Science.gov (United States)

    Peacock, Matthew; Brem, Reto; Macpherson, Peter; Karran, Peter

    2014-01-01

    Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage. PMID:25414333

  10. Action of some drugs on enzymes involved in DNA-repair and semiconservative DNA-synthesis

    International Nuclear Information System (INIS)

    Wawra, E.; Klein, W.; Kocsis, F.; Weniger, P.

    1975-07-01

    Different antirheumatic and cytostatic drugs had been tested by measurement of the thymidine incorporation into DNA of spleen cells under conditions, under which either DNA-synthesis or repair after gamma- or UV-irradiation takes place. There are substances, which inhibit either only the semiconservative DNA-synthesis (vinblastine, isonicotinic acid hydracide) or only DNA-repair after gamma-irradiation (mixture of penicillin-G and procaine-penicillin-G) or both (cyclophosphamide, phenylbutazone, procarbazine, nalidixic acid). Vincristine shows no effect on the thymidine incorporation in DNA, but by density gradient centrifugation it has been found that it influences the ligase reaction. Two DNA polymerases had been isolated from spleen cells, one of the low molecular and one of the high molecular weight type. The influences of the described drugs on these enzymes and on a deoxyribonuclease I from beef pancreas have been tested in ''in vitro'' systems. In all cases, it has been found that there is no effect or only a very small one, compared with the action of well known inhibitors as e.g. ethidium bromide and p-chloromercuribenzoate, and this cannot be responsible for the suppressions found in DNA-repair and semiconservative DNA-synthesis. (author)

  11. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

    Science.gov (United States)

    Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

    2016-01-08

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage binding protein.

    NARCIS (Netherlands)

    S. Keeney; A.P.M. Eker (André); T. Brody; W. Vermeulen (Wim); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan); S. Linn

    1994-01-01

    textabstractCells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells

  13. Analysis of a FANCE Splice Isoform in Regard to DNA Repair.

    Science.gov (United States)

    Bouffard, Frédérick; Plourde, Karine; Bélanger, Simon; Ouellette, Geneviève; Labrie, Yvan; Durocher, Francine

    2015-09-25

    The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of NtAI forecast a better initial response. We found an inverse relationship between BRCA1 expression and NtAI in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation...... of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (NtAI) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients...... also benefit from these agents. NtAI, a genomic measure of unfaithfully repaired DNA, may identify cancer patients likely to benefit from treatments targeting defective DNA repair. Cancer Discov; 2(4); 366–75. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 288...

  15. 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

  16. Cyclin A2 promotes DNA repair in the brain during both development and aging.

    Science.gov (United States)

    Gygli, Patrick E; Chang, Joshua C; Gokozan, Hamza N; Catacutan, Fay P; Schmidt, Theresa A; Kaya, Behiye; Goksel, Mustafa; Baig, Faisal S; Chen, Shannon; Griveau, Amelie; Michowski, Wojciech; Wong, Michael; Palanichamy, Kamalakannan; Sicinski, Piotr; Nelson, Randy J; Czeisler, Catherine; Otero, José J

    2016-07-01

    Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation inCCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice.

  17. Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius.

    Science.gov (United States)

    Torgasheva, Natalya A; Menzorova, Natalya I; Sibirtsev, Yurii T; Rasskazov, Valery A; Zharkov, Dmitry O; Nevinsky, Georgy A

    2016-06-21

    In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

  18. Chromatin associated mechanisms in base excision repair - nucleosome remodeling and DNA transcription, two key players.

    Science.gov (United States)

    Menoni, Hervé; Di Mascio, Paolo; Cadet, Jean; Dimitrov, Stefan; Angelov, Dimitar

    2017-06-01

    Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin? Copyright © 2017 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  20. DNA repair capacity in the rat respiratory tract

    International Nuclear Information System (INIS)

    Bond, J.A.; Gubin, J.M.; Johnson, N.F.

    1988-01-01

    A product of alkylating agents and DNA, O 6 -methylguanine, can mispair with thymine, resulting in initiation of a carcinogenic tissue response. O 6 -alkylguanine-DNA alkyltransferase (AGT) is an acceptor protein responsible for repairing O 6 -methylguanine. The purpose of our experiments was to characterize AGT activity in vitro in tissue and cell extracts of the respiratory tract, a target tissue for inhaled alkylating agents. Removal of [ 3 H]Methyl from O 6 -methylguanine was measured by high-pressure liquid chromatography after incubation of tissue and cell extracts with the [ 3 H]DNA. With the exception of tracheal and bronchial extracts, all tissues and cells analyzed contained AGT activity, which increased in proportion to the amount of protein added to reaction flasks. AGT activity in tracheal and bronchial extracts was only detected at the highest protein concentration used (1.5 mg protein/mL) and ranged from 10-15 fmole/mg protein. AGT activity in the respiratory tract was highest in the lung and a region of the nasal tissue (i.e., ethmoturbinates) and ranged from 45-75 fmole/mg protein. These data suggest that methylated DNA in specific regions of the rat respiratory tract should be readily repaired, albeit to different extents. (author)

  1. Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

    International Nuclear Information System (INIS)

    Yoshimoto, Koji; Mizoguchi, Masahiro; Hata, Nobuhiro; Murata, Hideki; Hatae, Ryusuke; Amano, Toshiyuki; Nakamizo, Akira; Sasaki, Tomio

    2012-01-01

    Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ) is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma (GBM). Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been described as the main modulator to determine the sensitivity of GBM to TMZ, a subset of GBM does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR), and the base excision repair (BER) pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break repair and double-strand break repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.

  2. Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimoto, Koji; Mizoguchi, Masahiro; Hata, Nobuhiro; Murata, Hideki; Hatae, Ryusuke; Amano, Toshiyuki; Nakamizo, Akira; Sasaki, Tomio, E-mail: kyoshimo@ns.med.kyushu-u.ac.jp [Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka (Japan)

    2012-12-05

    Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ) is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma (GBM). Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been described as the main modulator to determine the sensitivity of GBM to TMZ, a subset of GBM does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR), and the base excision repair (BER) pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break repair and double-strand break repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.

  3. Absence of specificity in inhibition of DNA repair replication by DNA-binding agents, cocarcinogens, and steroids in human cells

    International Nuclear Information System (INIS)

    Cleaver, J.E.; Painter, R.B.

    1975-01-01

    Although many chemicals, including cocarcinogens, DNA-binding agents, and steroids, inhibit repair replication of ultraviolet-induced damage to DNA in human lymphocytes and proliferating cells in culture, none of these chemicals is specific. Our results show that all the chemicals we tested inhibit normal DNA synthesis as much as or more than they inhibit repair replication. There is thus no evidence in our results to support the hypothesis that cocarcinogens are specific inhibitors of DNA repair or that any of the chemicals studied might be useful adjuncts to tumor therapy merely because of specific inhibition of radiation repair mechanisms

  4. The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA.

    Science.gov (United States)

    Rand, Lucinda; Hinds, Jason; Springer, Burkhard; Sander, Peter; Buxton, Roger S; Davis, Elaine O

    2003-11-01

    In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.

  5. Phytochemicals radiosensitize cancer cells by inhibiting DNA repair

    International Nuclear Information System (INIS)

    Singh, Rana P.

    2017-01-01

    Solid tumors are mostly treated with radiotherapy. Radiotherapy is toxic to normal tissues and also promote the invasiveness and radioresistance in cancer cells. The resistance against radiotherapy and adverse effects to normal cells reduce the overall therapeutic effects of the treatment. Radiosensitizing agents usually show limited success during clinical trials. Therefore, the search and development of new radiosensitizers showing selective response to only cancer cells is desirable. We analyzed the radiosensitizing effects including cell death effect of silibinin, a phytochemical on prostate cancer cells. Silibinin enhanced gamma radiation (2.5-10 Gy) induced inhibition in colony formation selectively in prostate cancer cells. In cell cycle progression, G2/M phase is the most sensitive phase for radiation-induced damage which was delayed by the compound treatment in radiation exposed cells. The lower concentrations of silibinin substantially enhanced radiation-induced apoptosis. A prolonged reactive oxygen species production was also observed in these treatments EGFR signaling pathway can contribute to radiation-induced pro-survival mechanisms and to the therapeutic resistance. Agent treatment reduced the IR-induced EGFR phosphorylation and consequently reversed the resistance mediating mechanisms within the cancer cell. Thus, inhibiting DNA repair in cancer cells would enhance therapeutic response of radiation in cancer cells. Silibinin affected the localization of EGFR and DNA-dependent protein kinase, the DNA-PK is known to be an important mediator of DSB repair in human cells, and showed increased number of pH2AX (ser139) foci, and thus indicating lower DNA repair in these cancer cells. This was also confirmed in the tumor xenograft study. Our findings suggest that a combination of silibinin with radiation could be an effective treatment of radioresistant human prostate cancer and warrants further investigation. (author)

  6. Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair

    International Nuclear Information System (INIS)

    Oliveri, D.R.

    1989-01-01

    The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens

  7. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

    2007-02-07

    The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

  8. Cloning and characterization of human DNA repair genes

    International Nuclear Information System (INIS)

    Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Stewart, S.A.; Carrano, A.V.

    1987-01-01

    The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

  9. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Hammond, R.A.; Miller, M.R.; McClung, J.K.

    1990-01-01

    The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

  10. Reciprocal Regulation between DNA-PKcs and Snail1 Conferring Genomic Instability

    International Nuclear Information System (INIS)

    Seo, Haeng Ran; Lee, Hae June; Jin, Yeung Bae; Bae, Sang Woo; Lee, Yun Sil; Kim, Nam Hee; Kim, Hyun Sil; Nam, Hyung Wook; Yook, Jong In

    2010-01-01

    Although the roles of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) involving non-homologous end joining (NHEJ) of DNA repair are well recognized, the biological mechanisms and regulators by which DNA-PKcs regulate genomic instability are not clearly defined. We show herein that DNA-PKcs activity resulting from DNA damage caused by ionizing radiation (IR) phosphorylates Snail1 at serine 100, which results in increased Snail1 expression and its function by inhibition of GSK-3-mediated phosphorylation. Furthermore, Snail1 phosphorylated at serine 100 can reciprocally inhibit kinase activity of DNA-PKcs, resulting in an inhibition to recruit DNA-PKcs or Ku70/80 to a DNA double-strand break site, and ultimately inhibition of DNA repair activity. The impairment of repair activity by a direct interaction between Snail1 and DNA-PKcs increases the resistance to DNA damaging agents, such as IR, and genomic instability. Our findings provide a novel cellular mechanism for induction of genomic instability by reciprocal regulation of DNA-PKcs and Snail1

  11. DNA repair in a Fanconi's anemia fibroblast cell strain

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

    1979-01-01

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

  12. DNA repair in a Fanconi's anemia fibroblast cell strain

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

    1979-01-26

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

  13. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

  14. 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

  15. DNA repair and its relation to recombination-deficient and other mutations in Bacillus subtilis

    International Nuclear Information System (INIS)

    Ganesan, A.T.

    1975-01-01

    DNA repair processes operating in Bacillus subtilis are similar to other transformable bacterial systems. Radiation-sensitive, recombination-deficient mutants are blocked in distinct steps leading to recombination. DNA polymerase I is essential for the repair of x-ray-induced damage to DNA but not for recombination

  16. Physical interaction between components of DNA mismatch repair and nucleotide excision repair

    International Nuclear Information System (INIS)

    Bertrand, P.; Tishkoff, D.X.; Filosi, N.; Dasgupta, R.; Kolodner, R.D.

    1998-01-01

    Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as 'bait,' and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes

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

    International Nuclear Information System (INIS)

    Charp, P.A.

    1987-01-01

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

  18. Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

    International Nuclear Information System (INIS)

    Mullenders, L.H.F.; Kesteren, A.C. van; Bussmann, C.J.M.; Zeeland, A.A. van; Natarajan, A.T.

    1984-01-01

    The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimer-specific endonuclease V of bacteriophage T 4 . The results suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts the authors observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in regions of the genome. (Auth.)

  19. Conformational Analysis of DNA Repair Intermediates by Time-Resolved Fluorescence Spectroscopy

    OpenAIRE

    Lin, Su; Horning, David P.; Szostak, Jack W.; Chaput, John C.

    2009-01-01

    DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NM...

  20. 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.

  1. Cell sensitivity to irradiation and DNA repair processes. II

    International Nuclear Information System (INIS)

    Kozubek, S.; Krasavin, E.A.

    1984-01-01

    A new model of DNA single-strand break (SSB) and double-strand break (DSB) induction by radiations of different linear energy transfer (LET) has been developed. Utilizing quadratic dependence of the dose that delta-electrons depart in the track of heavy particles the fraction of heavy particle energy deposited in the target of DNA dimensions has been calculated. SSBs arise from energy depositions in one strand of DNA, direct DSBs arise from two SSBs on opposite strands of DNA in the track of one particle. It is concluded that DSB's induced by γ-radiation are mostly of enzymatic origin, meanwhile DSB's induced by high-LET radiation are direct DSB's. The dependence of radiosensitivity D 0 -1 on LET (L) for isogenic mutants of E. coli with different sensitivity to γ-radiation has been determined on the bases of the model and considering microscopic energy fluctuations. The shape of D 0 -1 (L) function is formed both by physical characteristics of radiation and by the ability of cells to repair some types of DNA damage. The model provides a basis for further investigation. (author)

  2. DNA repair by the Ada protein of E. coli

    International Nuclear Information System (INIS)

    Karran, P.; Hall, J.

    1988-01-01

    This paper discusses the Ada protein of E. coli which exemplifies the highly specialized nature of the enzymes which have evolved to repair DNA. According to the authors, this protein exhibits not only novel mechanistic features but also provides an apparently unique example of a strategy for controlling gene expression in E. coli. They report that knowledge of the properties and mode of action of the Ada protein has afforded insight into how human cells are affected by alkylating agents, including those used in chemotherapy

  3. Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks

    International Nuclear Information System (INIS)

    Turney, Benjamin W.; Kerr, Martin; Chitnis, Meenali M.; Lodhia, Kunal; Wang, Yong; Riedemann, Johann; Rochester, Mark; Protheroe, Andrew S.; Brewster, Simon F.; Macaulay, Valentine M.

    2012-01-01

    Background and purpose: IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. Methods: We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. Results: We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30–40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. Conclusions: These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments.

  4. Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks.

    Science.gov (United States)

    Turney, Benjamin W; Kerr, Martin; Chitnis, Meenali M; Lodhia, Kunal; Wang, Yong; Riedemann, Johann; Rochester, Mark; Protheroe, Andrew S; Brewster, Simon F; Macaulay, Valentine M

    2012-06-01

    IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30-40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  5. FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

    International Nuclear Information System (INIS)

    Marie, Melanie; Hafner, Sophie; Moratille, Sandra; Vaigot, Pierre; Rigaud, Odile; Martin, Michele T.; Mine, Solene

    2012-01-01

    Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2. (authors)

  6. Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

    Directory of Open Access Journals (Sweden)

    Yang Ching-Hsiu

    2008-09-01

    Full Text Available Abstract Background Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2 and DNA glycosylase (Ogg1, MutY. Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression. Results Our results show that hepatic steatosis was induced by a high-fat diet and improved by adding pioglitazone. Malondialdehyde concentration and 8-oxoG staining were strongly increased in the high-fat diet group, but attenuated by pioglitazone. Gene expressions of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY significantly decreased in the high-fat diet group but reversed by pioglitazone co-administration. Conclusion The attenuation of hepatic oxidative DNA damage by pioglitazone in a high-fat diet may be mediated by up-regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The diminution of oxidative damage may explain the clinical benefit of pioglitazone treatment in patients with non-alcoholic fatty liver disease.

  7. Pioglitazone retrieves hepatic antioxidant DNA repair in a mice model of high fat diet

    Science.gov (United States)

    Hsiao, Pi-Jung; Hsieh, Tusty-Jiuan; Kuo, Kung-Kai; Hung, Wei-Wen; Tsai, Kun-Bow; Yang, Ching-Hsiu; Yu, Ming-Lung; Shin, Shyi-Jang

    2008-01-01

    Background Pioglitazone was reported to improve hepatic steatosis and necroinflammation in human studies. To investigate whether the hepato-protective effect of pioglitazone was associated with an improvement of antioxidant defense mechanism, oxidative DNA damage and repair activity were determined in a high fat diet model. Male C57BL/6 mice were respectively fed with a 30% fat diet, the same diet with pioglitazone 100 mg/kg/day, or a chow diet as control for 8 weeks. Tissue oxidative stress was indicated by malondialdehyde concentration. Oxidative DNA damage was detected by immunohistochemical 8-oxoG staining. Enzymatic antioxidant defense was detected by the real-time PCR of superoxide dismutase (Sod1, Sod2) and DNA glycosylase (Ogg1, MutY). Oxidative DNA repair was detected by immunohistochemical staining and western blotting of OGG1 expression. Results Our results show that hepatic steatosis was induced by a high-fat diet and improved by adding pioglitazone. Malondialdehyde concentration and 8-oxoG staining were strongly increased in the high-fat diet group, but attenuated by pioglitazone. Gene expressions of antioxidant defense mechanism: Sod1, Sod2, Ogg1 and MutY significantly decreased in the high-fat diet group but reversed by pioglitazone co-administration. Conclusion The attenuation of hepatic oxidative DNA damage by pioglitazone in a high-fat diet may be mediated by up-regulation of the antioxidant defense mechanism and oxidative DNA repair activity. The diminution of oxidative damage may explain the clinical benefit of pioglitazone treatment in patients with non-alcoholic fatty liver disease. PMID:18822121

  8. 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

  9. Organizing DNA repair in the nucleus: DSBs hit the road.

    Science.gov (United States)

    Marnef, Aline; Legube, Gaëlle

    2017-06-01

    In the past decade, large-scale movements of DNA double strand breaks (DSBs) have repeatedly been identified following DNA damage. These mobility events include clustering, anchoring or peripheral movement at subnuclear structures. Recent work suggests roles for motion in homology search and in break sequestration to preclude deleterious outcomes. Yet, the precise functions of these movements still remain relatively obscure, and the same holds true for the determinants. Here we review recent advances in this exciting area of research, and highlight that a recurrent characteristic of mobile DSBs may lie in their inability to undergo rapid repair. A major future challenge remains to understand how DSB mobility impacts on genome integrity. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Radiation induced DNA damage and repair in mutagenesis

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  11. Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase.

    NARCIS (Netherlands)

    B.P. Engelward (Bevin); G. Weeda (Geert); M.D. Wyatt; J.L.M. Broekhof (Jose'); J. de Wit (Jan); I. Donker (Ingrid); J.M. Allan (James); B. Gold (Bert); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1997-01-01

    textabstract3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA

  12. Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates, chromosomal aberrations and single-strand breaks in DNA

    Czech Academy of Sciences Publication Activity Database

    Vodička, Pavel; Kumar, R.; Štětina, R.; Sanyal, S.; Souček, P.; Haufroid, V.; Dušinská, M.; Kuricová, M.; Zámečníková, M.; Musak, L.; Buchancová, J.; Norppa, H.; Hirvonen, A.; Vodičková, L.; Naccarati, Alessio; Matoušů, Zora; Hemminki, K.

    2004-01-01

    Roč. 25, č. 5 (2004), s. 757-763 ISSN 0143-3334 R&D Projects: GA ČR GA310/03/0437; GA ČR GA310/01/0802 Institutional research plan: CEZ:AV0Z5039906 Keywords : DNA repair rates * genotoxicity Subject RIV: FM - Hygiene Impact factor: 5.375, year: 2004

  13. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  14. 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)

  15. DNA-membrane complex restoration in Micrococcus radiodurans after X-irradiation: relation to repair, DNA synthesis and DNA degradation

    Energy Technology Data Exchange (ETDEWEB)

    Dardalhon-Samsonoff, M; Averbeck, D [Institut du Radium, 75 - Paris (France). Lab. Curie

    1980-07-01

    The DNA-membrane complex in Micrococcus radiodurans was shown to be essentially constituted of proteins, lipids and DNA. The complex was dissociated immediately after X-irradiation of cells and restored during post-incubation in complete medium. In X-irradiated protoplasts some DNA remained associated with the complex. Restoration of the complex during post-incubation was only seen in a medium favouring DNA polymerase and ligase activities. Under this condition no DNA synthesis occurred, suggesting that complex restoration may involve ligase activity. The complex restoration in the wild type and the X-ray sensitive mutant UV17 of M. radiodurans was strictly dependent on the X-ray dose. It was correlated with survival and DNA degradation but always preceded the onset of DNA synthesis after X-irradiation. At the same dose the complex restoration was about 2 fold lower in mutant than in wild type cells indicating that the restoration of the complex is related to repair capacity. The results are consistent with the idea that the complex protects X-irradiated DNA of M. radiodurans from further breakdown and, subsequently, permits DNA synthesis and repair to occur.

  16. Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

    International Nuclear Information System (INIS)

    Yatani, Ryuichi; Tohgo, Yukihiro; Kunishima, Nobuyoshi.

    1975-01-01

    UV (ultraviolet) damaged DNA and its repair of various cultured animal cells were observed by radioimmunoassay using anti-serum against the UV irradiation induced heat-degenerated DNA. There is some difference among the cells of used animals according to their DNA repairabilities. The cells were divided into four groups according to the existence or strength of their repairabilities. 1) excision repair type: cells of men and chimpanzees. 2) photoreactivation type: cells derived from Tachydromus tachydromoides and chicks. 3) photoreactivation with excision repair: cells of rats, kangaroos and mosquitos. 4) non-excision repair type: cells of mice, Meriones and rats. Animal cells have plural types of repair. Main types of repair will differ according to the kind of animals. (Ichikawa, K.)

  17. Genetic polymorphisms in 85 DNA repair genes and bladder cancer risk.

    Science.gov (United States)

    Michiels, Stefan; Laplanche, Agnès; Boulet, Thomas; Dessen, Philippe; Guillonneau, Bertrand; Méjean, Arnaud; Desgrandchamps, François; Lathrop, Mark; Sarasin, Alain; Benhamou, Simone

    2009-05-01

    Several defense mechanisms have been developed and maintained during the evolution to protect human cells against damage produced from exogenous or endogenous sources. We examined the associations between bladder cancer and a panel of 652 polymorphisms from 85 genes involved in maintenance of genetic stability [base excision repair, nucleotide excision repair, double-strand break repair (DSBR) and mismatch repair, as well as DNA synthesis and cell cycle regulation pathways] in 201 incident bladder cancer cases and 326 hospital controls. Score statistics were used to test differences in haplotype frequencies between cases and controls in an unconditional logistic regression model. To account for multiple testing, we associated to each P-value the expected proportion of false discoveries (q-value). Haplotype analysis revealed significant associations (P genes (POLB and FANCA) with an associated q-value of 24%. A permutation test was also used to determine whether, in each pathway analyzed, there are more variants whose allelic frequencies are different between cases and controls as compared with what would be expected by chance. Differences were found for cell cycle regulation (P = 0.02) and to a lesser extent for DSBR (P = 0.05) pathways. These results hint to a few potential candidate genes; however, our study was limited by the small sample size and therefore low statistical power to detect associations. It is anticipated that genome-wide association studies will open new perspectives for interpretation of the results of extensive candidate gene studies such as ours.

  18. In TFIIH, XPD helicase is exclusively devoted to DNA repair.

    Directory of Open Access Journals (Sweden)

    Jochen Kuper

    2014-09-01

    Full Text Available The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER. Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

  19. Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yarosh, Daniel B

    2002-11-30

    The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

  20. SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Waaqo Daddacha

    2017-08-01

    Full Text Available DNA double-strand break (DSB repair by homologous recombination (HR is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.

  1. Association of DNA repair polymorphisms with DNA repair functional outcomes in healthy human subjects

    Czech Academy of Sciences Publication Activity Database

    Vodička, Pavel; Štětina, R.; Poláková, Veronika; Tulupová, Elena; Naccarati, Alessio; Vodičková, Ludmila; Kumar, R.; Hánová, Monika; Pardini, Barbara; Slyšková, Jana; Musak, L.; De Palma, G.; Souček, P.; Hemminki, K.

    2007-01-01

    Roč. 28, č. 3 (2007), s. 657-664 ISSN 0143-3334 R&D Projects: GA MZd NR8563; GA ČR GA310/05/2626 Institutional research plan: CEZ:AV0Z50390512 Keywords : Base excision DNA * Single-strand breaks * Peripheral blood lymphocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.406, year: 2007

  2. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  3. Analysis of DNA vulnerability to damage, repair and degradation in tissues of irradiated animals

    International Nuclear Information System (INIS)

    Ryabchenko, N.I.; Ivannik, B.P.

    1982-01-01

    Single-strand and paired ruptures of DNA were found to result in appearance of locally denaturated areas in its secondary structure and to disordered protein-DNA interaction. It was shown with the use of the viscosimeter method of measuring the molecular mass of single stranded high-polymeric DNA that cells of various tissues by the intensity of DNA repair can be divided into two groups, rapid- and slow-repair ones. Tissue specificity of enzyme function of the repair systems and systems responsible for post-irradiation DNA degradation depends on the activity of endonucleases synthesized by the cells both in health and in their irradiation-induced synthesis

  4. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18-83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...

  5. Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis

    Science.gov (United States)

    Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo

    2013-01-01

    SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

  6. Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

    Science.gov (United States)

    Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

    2016-09-01

    The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

  7. NAD+ Replenishment Improves Lifespan and Healthspan in Ataxia Telangiectasia Models via Mitophagy and DNA Repair.

    Science.gov (United States)

    Fang, Evandro Fei; Kassahun, Henok; Croteau, Deborah L; Scheibye-Knudsen, Morten; Marosi, Krisztina; Lu, Huiming; Shamanna, Raghavendra A; Kalyanasundaram, Sumana; Bollineni, Ravi Chand; Wilson, Mark A; Iser, Wendy B; Wollman, Bradley N; Morevati, Marya; Li, Jun; Kerr, Jesse S; Lu, Qiping; Waltz, Tyler B; Tian, Jane; Sinclair, David A; Mattson, Mark P; Nilsen, Hilde; Bohr, Vilhelm A

    2016-10-11

    Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by progressive neurodegeneration and cerebellar ataxia. A-T is causally linked to defects in ATM, a master regulator of the response to and repair of DNA double-strand breaks. The molecular basis of cerebellar atrophy and neurodegeneration in A-T patients is unclear. Here we report and examine the significance of increased PARylation, low NAD + , and mitochondrial dysfunction in ATM-deficient neurons, mice, and worms. Treatments that replenish intracellular NAD + reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD + also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial dysfunction through nuclear DNA damage-induced nuclear-mitochondrial signaling, and demonstrates that they are important pathophysiological determinants in premature aging of A-T, pointing to therapeutic interventions. Published by Elsevier Inc.

  8. Relationship of DNA repair and chromosome aberrations to potentially lethal damage repair in X-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Nagasawa, H.; Little, J.B.

    1980-01-01

    By the alkaline elution technique, the repair of x-ray-induced DNA single strand breaks and DNA-protein cross-links was investigated in stationary phase, contact-inhibited mouse cells. During the first hour of repair, approximately 90% of x-ray induced single strand breaks were rejoined whereas most of the remaining breaks were rejoined more slowly during the next 5 h. The number of residual non-rejoined single strand breaks was approximately proportional to the x-ray dose at early repair times. DNA-protein cross-links were removed at a slower rate - T 1/2 approximately 10 to 12 h. Cells were subcultured at low density at various times after irradiation and scored for colony survival, and chromosome aberrations in the first mitosis after sub-culture. Both cell lethality and the frequency of chromosome aberrations decreased during the first several hours of repair, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA strand breaks. The possible relationship of DNA repair to changes in survival and chromosome aberrations is discussed

  9. DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Inoue, Masao; Takebe, Hiraku.

    1978-01-01

    Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

  10. Guardians of the mycobacterial genome: A review on DNA repair systems in Mycobacterium tuberculosis.

    Science.gov (United States)

    Singh, Amandeep

    2017-12-01

    The genomic integrity of Mycobacterium tuberculosis is continuously threatened by the harsh survival conditions inside host macrophages, due to immune and antibiotic stresses. Faithful genome maintenance and repair must be accomplished under stress for the bacillus to survive in the host, necessitating a robust DNA repair system. The importance of DNA repair systems in pathogenesis is well established. Previous examination of the M. tuberculosis genome revealed homologues of almost all the major DNA repair systems, i.e. nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent developments in the field have pointed to the presence of novel proteins and pathways in mycobacteria. Homologues of archeal mismatch repair proteins were recently reported in mycobacteria, a pathway previously thought to be absent. RecBCD, the major nuclease-helicase enzymes involved in HR in E. coli, were implicated in the single-strand annealing (SSA) pathway. Novel roles of archeo-eukaryotic primase (AEP) polymerases, previously thought to be exclusive to NHEJ, have been reported in BER. Many new proteins with a probable role in DNA repair have also been discovered. It is now realized that the DNA repair systems in M. tuberculosis are highly evolved and have redundant backup mechanisms to mend the damage. This review is an attempt to summarize our current understanding of the DNA repair systems in M. tuberculosis.

  11. 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

  12. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    International Nuclear Information System (INIS)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A.

    2015-01-01

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer

  13. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A., E-mail: lsamant@uab.edu [Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, WTI320D, 1824 6th Avenue South, Birmingham, AL 35233 (United States)

    2015-07-21

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

  14. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    Science.gov (United States)

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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.

  16. DNA repair and DNA synthesis in leukemic and virus infected cells

    International Nuclear Information System (INIS)

    Tuschl, H.; Altmann, H.; Kovac, R.; Topaloglou, A.; Stacher, A.; Fanta, D.

    1978-09-01

    Autoradiographic determinations of unscheduled DNA synthesis in peripheral lymphocytes of leukemic patients showed strongly different results according to various types of disease of different forms of therapy, respectively. Similar investigations performed with lymphocytes of Herpes simplex infected persons during symptom-free intervals revealed imbalances of the repair system caused by virus infection. BND cellulose chromatography and measurement of 3 H-thymidine incorporation into single- and double stranded DNA fractions showed an increase in velocity of the rejoining process, but a decrease in total incorporation. Because of these results and the demonstration of the supercoiled structure of DNA it is suggested that virusinfections cause a faster rejoining of gaps, but at the same time leave a number of failures within DNA unrecognized. (author)

  17. Formation and repair of DNA-protein cross-links (DPCs) in newly replicated DNA

    International Nuclear Information System (INIS)

    Chiu, S.; Friedman, L.R.; Oleinick, N.L.

    1987-01-01

    DPCs preferentially involve proteins of the nuclear matrix, the site of replication and transcription. To elucidate the relationship with replication, the formation and repair of DPCs has been studied in newly replicated DNA. Log-phase V79 cells were pulsed with /sup 3/H-TdR (10-20 μCi/ml) for 30-90 sec at 22 0 followed by up to a 60 min chase at 37 0 . Irradiation (0-100 Gy) immediately after the pulse increases the labeled DNA in DPCs with a dose-dependence that is unaffected by the initial level of labeled DPC or by chase time. When cells are irradiated before the pulse, DNA synthesis is inhibited; however, release of pulse-labeled DPCs appears normal. The data suggest that during replication, DNA is cross-linked to (matrix) protein, contributing to background DPCs

  18. DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

    Full Text Available PURPOSE: In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. METHODS AND MATERIALS: In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. RESULTS: Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. CONCLUSIONS: Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

  19. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng [National Yang Ming Univ., Taipei, Taiwan (China). Inst. of Radiological Sciences] (and others)

    2001-12-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

  20. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    International Nuclear Information System (INIS)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng

    2001-01-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

  1. Use of hydroxyurea in the measurement of DNA repair by the BND cellulose method

    International Nuclear Information System (INIS)

    Irwin, J.; Strauss, B.

    1980-01-01

    Hydroxyurea inhibition is a convenient method of suppressing replicative DNA synthesis for DNA excision-repair measurement by the BND cellulose technique. Nonetheless, hydroxyurea can introduce artefacts by direct reaction with repair-inducing compounds and by long-term inhibition of the overall repair process. A simple technique of overcoming these problems is described. Cells are reacted with repair-inducing compounds in the absence of hydroxyurea, the cells are washed free of inducer, hydroxyurea is added to 2 mM, and after a short period to establish replication inhibition, 3 H dThd is added and repair measured over a one-hour incubation period

  2. The DNA repair capability of cdc9, the saccharomyces cerevisiae mutant defective in DNA ligase

    International Nuclear Information System (INIS)

    Johnston, L.H.

    1979-01-01

    The cell cycle mutant, cdc9, in the yeast Saccharomyces cerevisiae is defective in DNA ligase with the consequence to be deficient in the repair of DNA damaged by methyl methane sulphonate. On the other hand survival of cdc9 after irradiation by γ-rays is little different from that of the wild-type, even after a period of stress at the restrictive temperature. The mutant cdc9 is not allelic with any known rad or mms mutants. (orig./AJ) [de

  3. 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.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  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. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    International Nuclear Information System (INIS)

    Canuto, K S; Sergio, L P S; Marciano, R S; Guimarães, O R; Polignano, G A C; Geller, M; Fonseca, A S; Paoli, F

    2013-01-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase. (letter)

  7. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Karen L.; Dashner, Erica J. [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Tsosie, Ranalda [Department of Chemistry and Biochemistry, University of Montana, Missoula, MT 59812 (United States); Cho, Young Mi [Department of Food and Nutrition, College of Human Ecology, Hanyang University, Seoul 133-791 (Korea, Republic of); Lewis, Johnnye [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Community Environmental Health Program, University of New Mexico Health Sciences Center College of Pharmacy, Albuquerque, NM 87131 (United States); Hudson, Laurie G., E-mail: lhudson@salud.unm.edu [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States)

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.

  8. DNA replication and repair in Tilapia cells. 1. The effect of ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yew, F.H.; Chang, L.M. (National Taiwan Univ., Taipei (China))

    1984-12-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-..beta..-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor.

  9. 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.

  10. Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis

    NARCIS (Netherlands)

    E.J. Uringa

    2005-01-01

    textabstractMy PhD project encompassed studies on the functions of several different proteins, all involved in DNA repair, in somatic and germ-line cells. Hr6b and Rad18Sc are involved in a DNA repair mechanism called ‘Replicative Damage Bypass’ (RDB), and function as ubiquitin conjugating

  11. Some important advances in DNA repair study on the mammalian cells

    International Nuclear Information System (INIS)

    Xia Shouxuan.

    1991-01-01

    In the recent years the study of DNA damage and repair in the mammalian cells has gone deeply at gene level and got the following advances: (1) For a long time DNA has been considered to be an uniform unit in case of damage and repair. Now this concept should be replaced by the non-random distribution of damage and heterogenous repair in the genome. These would allow us to study cellular mutagenesis, carcinogenesis, aging and dying processes in great detail, and would be beneficial to the elucidation of mechanisms of radiation sickness and chemical toxicology. (2) The advent of new techniques in molecular biology has made it possible to isolate and clone the human DNA repair genes. Up to now more than ten human DNA repair genes have been cloned and these works would have an important impact on the theoretical and practical study in this field. Because DNA repair system is very complicate, voluminous work should be done in the future. (3) The technique of gene transfer has been efficiently used in the study of DNA repair in mammalian cells and has made great contribution in the cellular engineering. It could modify the genetic behavior of the gene-accepting cells, and enhance the DNA repair ability to physical and chemical damages. Human gene therapy for DNA deficient diseases is now on the day

  12. RTEL1 contributes to DNA replication and repair and telomere maintenance

    NARCIS (Netherlands)

    Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A.; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M.

    2012-01-01

    Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance

  13. Repetitious nature of repaired DNA in mammalian cells. Progress report, June 1, 1976--February 28, 1977

    International Nuclear Information System (INIS)

    Meltz, M.L.

    1977-02-01

    Progress is reported on studies of DNA repair in cultured mouse L fibroblasts, human diploid fibroblasts, and cultured human lymphoblastoid cell lines. Data are included on the effects of methyl methanesulfonate treatment, uv light, and age of cell donors on repair replication of DNA

  14. RTEL1 contributes to DNA replication and repair and telomere maintenance

    NARCIS (Netherlands)

    E.J. Uringa; K. Lisaingo (Kathleen); H.A. Pickett (Hilda); J. Brind'Amour (Julie); J.-H. Rohde (Jan-Hendrik); A. Zelensky (Alexander); J. Essers (Jeroen); P.M. Lansdorp (Peter)

    2012-01-01

    textabstractTelomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere

  15. Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite

    International Nuclear Information System (INIS)

    Sykora, Peter; Snow, Elizabeth T.

    2008-01-01

    Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase β (Pol β) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol β and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 μM. However, at lower doses Pol β mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol β was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis

  16. Genetic variation in DNA repair gene XRCC7 (G6721T) and susceptibility to breast cancer.

    Science.gov (United States)

    Nasiri, Meysam; Saadat, Iraj; Omidvari, Shahpour; Saadat, Mostafa

    2012-08-15

    The human XRCC7 is a DNA double-strand break (DSBs) repair gene, involved in non-homologous end joining (NHEJ). It is speculated that DNA DSBs repair have an important role during development of breast cancer. The human XRCC7 is a NHEJ DSBs repair gene. Genetic variation G6721T of XRCC7 (rs7003908) is located in the intron 8 of the gene. This polymorphism may regulate splicing and cause mRNA instability. In the present study, we specifically investigated whether common G6721T genetic variant of XRCC7 was associated with an altered risk of breast cancer. The present study included 362 females with breast cancer. Age frequency-matched controls (362 persons) were randomly selected from the healthy female blood donors, according to the age distribution of the cases. Using RFLP-PCR based method, the polymorphism of XRCC7 was determined. The TG (OR=1.20, 95% CI: 0.83-1.74, P=0.320) and TT (OR=1.01, 95% CI: 0.67-1.53, P=0.933) genotypes had no significant effect on risk of breast cancer, in comparison with the GG genotype. Our present findings indicate that the TT and TG genotypes were not associated with an altered breast cancer risk. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Repair replication in replicating and nonreplicating DNA after irradiation with uv light

    Energy Technology Data Exchange (ETDEWEB)

    Slor, H.; Cleaver, J.E.

    1978-06-01

    Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 h of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.

  18. Reduced DNA repair in mouse satellite DNA after treatment with methylmethanesulfonate, and N-methyl-N-nitrosourea.

    Science.gov (United States)

    Bodell, W J; Banerjee, M R

    1976-01-01

    We have measured DNA repair in mouse satellite and main band DNA as resolved by Ag+-Cs2SO4 centrifugation in response to treatment with the alkylating agents, methyl methanesulfonate, and N-methyl-N-nitrosourea. We find that there is a statistically significant lower incorporation of 3H-Tdr into the satellite DNA as compared to the main band at varying periods after treatment with the alkylating agents. This suggests a reduced repair activity in the satellite DNA. We have measured the extent of binding of 14C-methyl methanesulfonate to the satellite, and main band DNA, and no difference in binding was observed, indicating that the reduced repair activity of satellite DNA is not due to a difference in binding of alkylating agents. We believe that the reduced incorporation of 3H-Tdr into satellite DNA may be due to its location in the condensed chromatin fraction. PMID:184436

  19. DNA Base Excision Repair (BER) and Cancer Gene Therapy: Use of the Human N-mythlpurien DNA Glycosylase (MPG) to Sensitize Breast Cancer Cells to Low Dose Chemotherapy

    National Research Council Canada - National Science Library

    Harvey, Tia

    2003-01-01

    The DNA Base Excision Repair (PER) pathway is responsible for the repair of alkylation and oxidative DNA damage resulting in protection against the deleterious effects of endogenous and exogenous agents encountered on a daily basis...

  20. Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

    OpenAIRE

    Tagua, Victor G.; Pausch, Marcell; Eckel, Maike; Gutiérrez, Gabriel; Miralles-Durán, Alejandro; Sanz, Catalina; Eslava, Arturo P.; Pokorny, Richard; Corrochano, Luis M.; Batschauer, Alfred

    2015-01-01

    DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryp- tochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B–induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair C...

  1. UV-inducible DNA repair in the cyanobacteria Anabaena spp

    International Nuclear Information System (INIS)

    Levine, E.; Thiel, T.

    1987-01-01

    Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation

  2. H4K20me0 marks post-replicative chromatin and recruits the TONSL–MMS22L DNA repair complex

    DEFF Research Database (Denmark)

    Saredi, Giulia; Huang, Hongda; Hammond, Colin M

    2016-01-01

    After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal...

  3. Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells

    International Nuclear Information System (INIS)

    Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

    2015-01-01

    Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage

  4. Repair of single-strand breaks induced in the DNA of Proteus mirabilis by excision repair after UV-irradiation

    International Nuclear Information System (INIS)

    Stoerl, K.; Mund, C.

    1977-01-01

    Single-strand breaks have been produced in the DNA of P. mirabilis after UV-irradiation in dependence on the incident UV-doses. It has been found that there exists a discrepancy between the single-strand breaks estimated from sedimentation in alkaline sucrose gradients and the expected single-strand breaks approximated from measurements of dimer excision. The low number in incision breaks observed by sedimentation experiments is an indication that the cells are able to repair the excision-induced breaks as fast as they are formed. Toluenized cells have been used for investigation of the incision step independently of subsequent repair processes. In presence of NMN the appearance of more single-strand breaks in the DNA has been observed. Furthermore, the number of incision breaks in toluenized cells increased in presence of exogenous ATP. The completion of the excision repair process has been investigated by observing the rejoining of incision breaks. After irradiation with UV-doses higher than approximately 240 erg/mm 2 the number of single-strand breaks remaining unrepaired in the DNA increased. Studies of the influence of nutrition conditions on the repair process have shown approximately the same capacity for repair of single-strand breaks in growth medium as well as in buffer. Progress in the excision repair was also followed by investigation of the DNA synthesized at the template-DNA containing the pyrimidine dimers. In comparison with E. coli, P. mirabilis showed a somewhat lower efficiency for the repair of single-strand breaks during the excision repair. (author)

  5. DNA single-strand breaks during repair of uv damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Kohn, K.W.; Kann, H.E. Jr.

    1976-01-01

    The method of DNA alkaline elution was applied to a study of the formation and resealing of DNA single-strand breaks after irradiation of human fibroblasts with ultraviolet light (UV). The general features of the results were consistent with current concepts of DNA excision repair, in that breaks appeared rapidly after uv, and resealed slowly in normal fibroblasts, whereas breaks did not appear in those cells of patients with xeroderma pigmentosum (XP) that are known to have defects in DNA repair synthesis. The appearance of breaks required a short post-uv incubation, consistent with the expected action of an endonuclease. Cells of the variant form of XP characterized by normal DNA repair synthesis exhibited normal production of breaks after uv, but were slower than normal cells in resealing these breaks. This difference was enhanced by caffeine. A model is proposed to relate this finding with a previously described defect in post-replication repair in these XP variant cells. DNA crosslinking appears to cause an underestimate in the measurement of DNA breakage after uv

  6. Relationship between DNA replication and DNA repair in human lymphocytes proliferating in vitro in the presence and in absence of mutagen

    International Nuclear Information System (INIS)

    Szyfter, K.; Wielgosz, M.Sz.; Kujawski, M.; Jaloszynski, P.; Zajaczek, S.

    1995-01-01

    The effects of mutagens on DNA replication and DNA repair were studied in peripheral blood lymphocytes (PBL) obtained from 21 healthy subjects, 2 samples from healthy heterozygote of ''Xeroderma pigmentosum'' (XP) and 2 samples from patient with clinically recognised XP. Inter-individual variations were found in DNA replication and in the level of spontaneous DNA repair measured under standard culture condition. Exposure of human PBL proliferating in vitro to B(a)P was followed by a partial inhibition of replicative DNA synthesis in all subjects and by an induction of DNA repair in healthy subjects. In XP patients DNA repair synthesis remained at the level attributed to spontaneous DNA repair. The response to mutagen varied individually. Results were analysed statistically. It was established that the studied indices of DNA synthesis correlate well with each other. The highest correlation was found between the levels of spontaneous and B(a)P-induced DNA repair. It is concluded that the level of spontaneous DNA repair is predictive for an estimation of cells ability to repair DNA damage. Inter-individual variations in the inhibition of DNA replication and in DNA repair synthesis are also dependent on the type of mutagen as shown by effects of other mutagens. Different effects of mutagen exposure on the inhibition of DNA replicative synthesis and induction of DNA repair can be explained by genetically controlled differences in the activity of enzymes responsible for mutagen processing and lesion removal. (author). 37 refs, 2 figs, 2 tabs

  7. DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection.

    Science.gov (United States)

    Gorna, Alina E; Bowater, Richard P; Dziadek, Jaroslaw

    2010-05-25

    Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens.

  8. Specificity and completeness of inhibition of DNA repair by novobiocin and aphidicolin

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1982-01-01

    Novobiocin and aphidicolin were both potent inhibitors of excision repair of u.v.-induced damage to DNA in human embryonic fibroblasts, and both also inhibited semiconservative DNA replication even more strongly. The mechanism of action of these two drugs is, however, different. Novobiocin inhibited repair replication without accumulating single-strand breaks, but aphidicolin inhibited repair replication with the accumulation of numerous single-strand breaks. Novobiocin appears to inhibit repair at an earlier stage than aphidicolin, which may indicate that DNA topoisomerases play a role in eukaryotic DNA repair. Digestion of DNA by exonuclease III indicated that repair patches in novobiocin-treated cells contained no excess 3'OH termini, whereas up to 40% of the repaired DNA in aphidicolin-treated cells had free 3'OH termini. Therefore, although aphidicolin resulted in the accumulation of single-strand breaks, many of the repair events escaped inhibition and the number of breaks is an underestimate of the true number of repair events

  9. DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA

    International Nuclear Information System (INIS)

    Pascucci, B.; Dogliotti, E.; Versteegh, A.; Hoffen, A. van; Zeeland, A.A. van; Mullenders, L.H.F.

    1997-01-01

    The induction and repair of DNA photolesions and mutations in the mitochondrial (mt) DNA of human cells in culture were analysed after cell exposure to UV-C light. The level of induction of cyclobutane pyrimidine dimers (CPD) in mitochondrial and nuclear DNA was comparable, while a higher frequency of pyrimidine (6-4) pyrimidone photoproducts (6-4 PP) was detected in mitochondrial than in nuclear DNA. Besides the known defect in CPD removal, mitochondria were shown to be deficient also in the excision of 6-4 PP. The effects of repair-defective conditions for the two major UV photolesions on mutagensis was assessed by analysing the frequency and spectrum of spontaneous and UV-induced mutations by restriction site mutation (RSM) method in a restriction endonuclease site, NciI (5'CCCGG3') located within the coding sequence of the mitochondrial gene for tRNA Leu . The spontaneous mutation frequency and spectrum at the NciI site of mitochondrial DNA was very similar to the RSM background mutation frequency (approximately 10 -5 ) and type (predominantly GC > AT transitions at GL 1 ) of the NciI site). Conversely, an approximately tenfold increase over background mutation frequency was recorded after cell exposure to 20 J/m 2 . In this case, the majority of mutations were C > T transitions preferentially located on the non-transcribed DNA strand at C 1 and C 2 of the NciI site. This mutation spectrum is expected by UV mutagenesis. This is the first evidence of induction of mutations in mitochondrial DNA by treatment of human cells with a carcinogen. (author)

  10. Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Ockey, C.H.

    1982-04-01

    DNA fibre autoradiography, after incorporation of high specific activity /sup 3/H-thymidine and /sup 3/H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) of G/sub 1/ (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G/sub 1/ cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.

  11. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    Science.gov (United States)

    Leem, S H; Ropp, P A; Sugino, A

    1994-08-11

    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

  12. Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability

    OpenAIRE

    Lambert, Muriel W

    2016-01-01

    Non-erythroid alpha spectrin (?IISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. ?IISp?s interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear ?IISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in ma...

  13. Repair capability of mammalian cell fractions demonstrated using infectivity of bacteriophage DNA

    International Nuclear Information System (INIS)

    Lai, S.P.; Lytle, C.D.; Benane, S.G.

    1976-01-01

    Extracts of Potoroo kidney cells (PtK2) were examined for ability to provide a repair function in vitro. The biological activity (infectivity) of uv-irradiated replicative form (RF) DNA of bacteriophage phiX174 was restored during incubation of the DNA with a nuclear extract but not with a cytoplasmic extract. The infectivity of the RF-DNA was determined in spheroplasts of E. coli C/sub s/, which is HCR - . This system for biological assay of uv-irradiated DNA repaired in vitro may be used to complement biochemical and biophysical investigations of molecular repair mechanisms in mammalian cells

  14. Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1979-01-01

    Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

  15. Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

    International Nuclear Information System (INIS)

    Stark, M.; Naiman, T.; Canaani, D.

    1989-01-01

    In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [ 3 H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line

  16. Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

    International Nuclear Information System (INIS)

    Clark, J.M.; Hanawalt, P.C.

    1982-01-01

    The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with 3 H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA

  17. Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

    Science.gov (United States)

    Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

    2016-05-26

    RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

  18. Concordant and opposite roles of DNA-PK and the "facilitator of chromatin transcription" (FACT in DNA repair, apoptosis and necrosis after cisplatin

    Directory of Open Access Journals (Sweden)

    Calkins Anne S

    2011-06-01

    Full Text Available Abstract Background Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK binds to DNA double strand breaks (DSBs through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. Results Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1, Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT. The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. Conclusions DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and

  19. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

    International Nuclear Information System (INIS)

    Cole, G.M.; Mortimer, R.K.

    1989-01-01

    The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae

  20. Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

    1991-09-01

    The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

  1. In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

    International Nuclear Information System (INIS)

    Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

    1988-01-01

    Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

  2. 1-{beta}-D-arabinofuranosylcytosine is cytotoxic in quiescent normal lymphocytes undergoing DNA excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori [Fukui Medical Univ., Matsuoka (Japan)

    2002-12-01

    We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-{beta}-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap-filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10{mu}M. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy. (author)

  3. 1-β-D-arabinofuranosylcytosine is cytotoxic in quiescent normal lymphocytes undergoing DNA excision repair

    International Nuclear Information System (INIS)

    Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori

    2002-01-01

    We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-β-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap-filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10μM. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy. (author)

  4. Impact of DNA mismatch repair system alterations on human fertility and related treatments.

    Science.gov (United States)

    Hu, Min-hao; Liu, Shu-yuan; Wang, Ning; Wu, Yan; Jin, Fan

    2016-01-01

    DNA mismatch repair (MMR) is one of the biological pathways, which plays a critical role in DNA homeostasis, primarily by repairing base-pair mismatches and insertion/deletion loops that occur during DNA replication. MMR also takes part in other metabolic pathways and regulates cell cycle arrest. Defects in MMR are associated with genomic instability, predisposition to certain types of cancers and resistance to certain therapeutic drugs. Moreover, genetic and epigenetic alterations in the MMR system demonstrate a significant relationship with human fertility and related treatments, which helps us to understand the etiology and susceptibility of human infertility. Alterations in the MMR system may also influence the health of offspring conceived by assisted reproductive technology in humans. However, further studies are needed to explore the specific mechanisms by which the MMR system may affect human infertility. This review addresses the physiological mechanisms of the MMR system and associations between alterations of the MMR system and human fertility and related treatments, and potential effects on the next generation.

  5. New polymorphisms of Xeroderma Pigmentosum DNA repair genes in myelodysplastic syndrome.

    Science.gov (United States)

    Santiago, Sabrina Pinheiro; Junior, Howard Lopes Ribeiro; de Sousa, Juliana Cordeiro; de Paula Borges, Daniela; de Oliveira, Roberta Taiane Germano; Farias, Izabelle Rocha; Costa, Marília Braga; Maia, Allan Rodrigo Soares; da Nóbrega Ito, Mayumi; Magalhães, Silvia Maria Meira; Pinheiro, Ronald Feitosa

    2017-07-01

    The association between Xeroderma Pigmentosum DNA repair genes (XPA rs1800975, XPC rs2228000, XPD rs1799793 and XPF rs1800067) polymorphisms and myelodysplastic syndrome (MDS) have not been reported. To assess the functional role between these polymorphisms and MDS, we evaluated 189 samples stratified in two groups: 95 bone marrow samples from MDS patients and 94 from healthy elderly volunteers used as controls. Genotypes for all polymorphisms were identified in DNA samples in an allelic discrimination experiment by real-time polymerase chain reaction (qPCR). We also studied the mRNA expression of XPA and XPC genes to evaluate if its polymorphisms were functional in 53 RNAm MDS patients by qPCR methodologies. To the rs2228000 polymorphism, the CT and TT polymorphic genotype were associated with increased odds ratio (OR) of more profound cytopenia (hemoglobin and neutrophils count). To the rs1799793 polymorphism, we found that the GG homozygous wild-type genotype was associated with a decreased chance of developing MDS. We observed low expression of XPA in younger patients, in hypoplastic MDS and patients with abnormal karyotype when presented AG or AA polymorphic genotypes. We also found that there was a statistically significant interaction between the presence of micromegakaryocyte on down regulation of XPC regarding the CT heterozygous genotype of the rs1800975 polymorphism. Our results suggest that new functional polymorphisms of Xeroderma Pigmentosum DNA repair genes in MDS are related to its pathogenesis and prognosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    International Nuclear Information System (INIS)

    Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl 2 ) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl 2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl 2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

  7. DNA-repair after UV-irradiation in skin fibroblasts from patients with actinic keratosis

    International Nuclear Information System (INIS)

    Sbano, E.; Andreassi, L.; Fimiani, M.; Valentino, A.; Baiocchi, R.

    1978-01-01

    Autoradiographic counting technique was utilized to measure the ultraviolet-induced unscheduled DNA synthesis of skin fibroblasts from 12 patients with chronic actinic keratosis and from 12 healthy donors of about the same age. In order to reveal a possible regional difference of DNA repair between the parts of the body ordinarily exposed and those parts unexposed to sunlight, two cell strains were used for each examined subject; one developed from the forehead skin and the other from the abdominal or axillary skin. Unscheduled DNA synthesis appeared depressed in actinic keratosis patients, as compared with controls. In all examined subjects, however, cell strains from exposed skin showed a DNA repair more active than cell strains from unexposed skin. These findings show that skin cancer may be promoted in actinic keratosis patients by a defect of DNA repair. The exalted DNA repair of chronically sun exposed skin is probably the consequence of a defensive process caused by enzymatic induction. (orig.) [de

  8. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  9. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    International Nuclear Information System (INIS)

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-01-01

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  10. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    International Nuclear Information System (INIS)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae

  11. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs

  12. Mutagenic DNA repair in escherichia coli. Pt. 6

    International Nuclear Information System (INIS)

    Bridges, B.A.

    1977-01-01

    In the non-filamenting tif-1 strain WP44sub(s)NF trp a dramatic enhancement of both UV and gamma ray mutability to Trp + was observed when irradiated bacteria were incubated on plates at 43 0 C. This enhanced mutability was progressively suppressed when the initial plating density exceeded 10 8 bacteria per plate and was not demonstrable in liquid media. Under optimal conditions more mutants were induced by gamma radiation than could reasonably be accounted for by the initial number of radiation-induced lesions in the DNA, implying the existence of some mechanism for amplifying the radiation effect. Moreover, the tif-enhanced mutation frequency could be obtained if incubation at restrictive temperature was delayed for up to 60 min in nutrient broth after irradiation, at a time when all known reparable DNA damage had been repaired and the number of viable bacteria had more than doubled. In plates the effect of high temperature was still fully demonstrable 120 min after irradiation. A compatible interpretation of the results would be that radiation causes a persisting physiological disturbance in the cell and that this enhances the spontaneous mutator effect occuring in tif-1 bacteria subjected to subsequent thermal shock. (orig./MG) [de

  13. Regulation Mechanism of HBV cccDNA

    Directory of Open Access Journals (Sweden)

    Cheng Jun

    2012-06-01

    Full Text Available Covalently closed circular (ccc DNA of hepatitis B virus (HBV existed in the nuclei of HBV infected hepatocytes with a half-life time of 14.3 years in a mathematic model. Viral protein feedback regulation in HBV life cycle to maintain vital viral replication is an important mechanism. Interleukin-6, epithelial growth factor, heme oxygenase-1, histones, and hepatocyte nuclear factors are demonstrated as the key regulators for HBV life cycle. CpG island structure and methylation status are involved in the regulation of HBV DNA replication. Nucleos(tide analogues are widely used in the clinical practice for the treatment of chronic hepatitis B patients, although no evidence indicating a direct inhibiton of HBV cccDNA. In the future, along with the study of HBV life cycle, new drugs including RNA interference technique, will pave the way to eliminate the HBV cccDNA from infected hepatocytes resulting final cure of chronic hepatitis B.

  14. Epigenetic regulation during fetal femur development: DNA methylation matters.

    Directory of Open Access Journals (Sweden)

    María C de Andrés

    Full Text Available Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs, adult chondrocytes and STRO-1(+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2 and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5--methyltransferase 1 (DNMT1 in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development

  15. The role of DNA base excision repair in brain homeostasis and disease

    DEFF Research Database (Denmark)

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah

    2015-01-01

    Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of p...... energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease....

  16. Energetics of DNA repair: effects of temperature on DNA repair in UV-irradiated peripheral blood leucocytes from chronic myeloid leukemic patients

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, A.; Sharma, R.; Jain, V.K.

    1988-05-01

    The effects of different temperatures (34-43/sup 0/C) were studied on the repair of UV-induced (254-nm) DNA damage and its energetics in peripheral blood leucocytes of chronic myeloid leukaemic patients. DNA repair was measured by the unscheduled DNA synthesis (UDS) technique. Cellular energy supply was modulated by inhibitors of oxidative phosphorylation (antimycin-A) and glycolysis (2-deoxy-D-glucose). It was observed that there is an increase in the amount of DNA repair with increasing temperatures up to 40/sup 0/C and a fall thereafter. Longer periods of heat treatment (4 h) beyond 40/sup 0/C were observed to further decrease the DNA repair. Increasing temperatures were observed to have no significant effect on the parameters of energy metabolism. Further, the activation energy of DNA repair was calculated as 92 +- 46 kJ/mol (22 +- 11 kcal/mol), which did not alter significantly even in the presence of inhibitors of energy metabolism.

  17. 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.

  18. Normal formation and repair of γ-radiation-induced single and double strand DNA breaks in Down syndrome fibroblasts

    International Nuclear Information System (INIS)

    Steiner, M.E.; Woods, W.G.

    1982-01-01

    Fibroblasts from patients with Down syndrome (Trisomy 21) were examined for repair capability of γ-radiation-induced single strand and double strand DNA breaks. Formation and repair of DNA breaks were determined by DNA alkaline and non-denaturing elution techniques. Down syndrome fibroblasts were found to repair single strand and double strand breaks as well as fibroblasts from normal controls. (orig.)

  19. The 2015 Nobel Prize in Chemistry The Discovery of Essential Mechanisms that Repair DNA Damage.

    Science.gov (United States)

    Lindahl, Tomas; Modrich, Paul; Sancar, Aziz

    2016-01-01

    The Royal Swedish Academy awarded the Nobel Prize in Chemistry for 2015 to Tomas Lindahl, Paul Modrich and Aziz Sancar for their discoveries in fundamental mechanisms of DNA repair. This pioneering research described three different essential pathways that correct DNA damage, safeguard the integrity of the genetic code to ensure its accurate replication through generations, and allow proper cell division. Working independently of each other, Tomas Lindahl, Paul Mo