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Sample records for damaged dna binding

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

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2016-02-01

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 Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs in a poly(ADP-ribose (PAR-dependent manner (unpublished data.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

  2. The DNA-binding box of human SPARTAN contributes to the targeting of Polη to DNA damage sites.

    Science.gov (United States)

    Toth, Agnes; Hegedus, Lili; Juhasz, Szilvia; Haracska, Lajos; Burkovics, Peter

    2017-01-01

    Inappropriate repair of UV-induced DNA damage results in human diseases such as Xeroderma pigmentosum (XP), which is associated with an extremely high risk of skin cancer. A variant form of XP is caused by the absence of Polη, which is normally able to bypass UV-induced DNA lesions in an error-free manner. However, Polη is highly error prone when replicating undamaged DNA and, thus, the regulation of the proper targeting of Polη is crucial for the prevention of mutagenesis and UV-induced cancer formation. Spartan is a novel regulator of the damage tolerance pathway, and its association with Ub-PCNA has a role in Polη targeting; however, our knowledge about its function is only rudimentary. Here, we describe a new biochemical property of purified human SPARTAN by showing that it is a DNA-binding protein. Using a DNA binding mutant, we provide in vivo evidence that DNA binding by SPARTAN regulates the targeting of Polη to damage sites after UV exposure, and this function contributes highly to its DNA-damage tolerance function. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Dutertre, Martin; Vagner, Stéphan

    2016-09-29

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

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

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

    2004-02-01

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

  5. DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

    DEFF Research Database (Denmark)

    Danielsen, Jannie Michaela Rendtlew; Povlsen, Lou Klitgaard; Villumsen, Bine Hare

    2012-01-01

    Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel......, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage-dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1...... at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8-Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together...

  6. Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

    Science.gov (United States)

    Miroshnikova, A D; Kuznetsova, A A; Kuznetsov, N A; Fedorova, O S

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

  7. Damaged DNA binding protein 2 plays a role in breast cancer cell growth.

    Directory of Open Access Journals (Sweden)

    Zilal Kattan

    Full Text Available The Damaged DNA binding protein 2 (DDB2, is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER-positive (MCF-7 and T47D, but not in ER-negative breast cancer (MDA-MB231 and SKBR3 or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold higher in ER-positive than in ER-negative tumor samples (P = 0.0208 from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase. These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

  8. Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.

    Science.gov (United States)

    Routledge, M N; Allan, J M; Garner, R C

    1997-07-01

    To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.

  9. Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1.

    Science.gov (United States)

    Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

    2017-04-04

    As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites.

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

    Science.gov (United States)

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

    2016-06-17

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

  11. Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

    Science.gov (United States)

    Bhaskaran, Natarajan; Gupta, Sanjay

    2014-01-01

    Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

  12. DNA damage and autophagy

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    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

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

  13. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Goto, Yamafumi [Department of Dermatology, Shinshu University School of Medicine, Matsumoto (Japan); Takata, Minoru [Department of Dermatology, Okayama University Graduate School of Medical Dentistry and Pharmaceutical Sciences, Okayama (Japan); Turkson, James; Li, Xiaoman Shawn [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Zervos, Antonis S., E-mail: azervos@mail.ucf.edu [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States)

    2011-01-07

    Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  14. The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions

    NARCIS (Netherlands)

    Moser, J; Volker, M; Kool, H; Alekseev, S; Vrieling, H; Yasui, A; van Zeeland, AA; Mullenders, LHF

    2005-01-01

    Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protei

  15. Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

    NARCIS (Netherlands)

    Alekseev, S.; Luijsterburg, M.S.; Pines, A.; Geverts, B.; Mari, P.-O.; Giglia-Mari, G.; Lans, H.; Houtsmuller, A.B.; Mullenders, L.H.F.; Hoeijmakers, J.H.J.; Vermeulen, W.

    2008-01-01

    Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged da

  16. Structural and mutational analyses of Deinococcus radiodurans UvrA2 provide insight into DNA binding and damage recognition by UvrAs.

    Science.gov (United States)

    Timmins, Joanna; Gordon, Elspeth; Caria, Sofia; Leonard, Gordon; Acajjaoui, Samira; Kuo, Mei-Shiue; Monchois, Vincent; McSweeney, Sean

    2009-04-15

    UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms. These structures, coupled with mutational analyses and comparison with the crystal structure of class I UvrA from Bacillus stearothermophilus, suggest a previously unsuspected role for the identified insertion domains of UvrAs in both DNA binding and damage recognition. Taken together, the available information suggests a model for how UvrA interacts with DNA and thus sheds new light on the molecular mechanisms underlying the role of UvrA in the early steps of NER.

  17. The DNA Damage-Binding Protein XPC Is a Frequent Target for Inactivation in Squamous Cell Carcinomas

    OpenAIRE

    de Feraudy, Sebastien; Ridd, Katie; Richards, Lauren M.; Kwok, Pui-Yan; Revet, Ingrid; Oh, Dennis; Feeney, Luzviminda; Cleaver, James E.

    2010-01-01

    XPC, the main damage-recognition protein responsible for nucleotide excision repair of UVB damage to DNA, is lost or mutated in xeroderma pigmentosum group C (XP-C), a rare inherited disease characterized by high incidence and early onset of non-melanoma and melanoma skin cancers. The high incidence of skin cancers in XP-C patients suggests that loss of expression of XPC protein might also provide a selective advantage for initiation and progression of similar cancers in non XP-C patients in ...

  18. A game of musical chairs: Pro- and anti-resection factors compete for TOPBP1 binding after DNA damage.

    Science.gov (United States)

    Shimada, Kenji; Gasser, Susan M

    2017-03-06

    DNA double strand breaks (DSBs) are generally repaired through nonhomologous end joining or homologous recombination. In this issue, Liu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607031) report that the conserved scaffold protein TOPBP1(Dpb11) provides binding sites for both pro- and anti-resection factors at DSBs, providing insights into repair pathway regulation. © 2017 Shimada and Gasser.

  19. Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Qianzheng; Battu, Aruna; Ray, Alo; Wani, Gulzar; Qian, Jiang; He, Jinshan; Wang, Qi-en [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Wani, Altaf A., E-mail: wani.2@osu.edu [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210 (United States); James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 (United States)

    2015-06-15

    Highlights: • HDAC1 and HDAC2 co-localize with UV radiation-induced DNA damage sites. • HDAC1 translocation to chromatin is dependent on DDB2 function. • HDAC1 and HDAC2 are involved in H3K56Ac deacetylation. • H3K56Ac deacetylation requires DDB1 and DDB2 but not XPA or XPC functions. • HDAC1/2 depletion decreases XPC ubiquitination and local γH2AX accumulation. - Abstract: Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

  20. DNA binding hydroxyl radical probes

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Vicky J.; Konigsfeld, Katie M.; Aguilera, Joe A. [Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610 (United States); Milligan, Jamie R., E-mail: jmilligan@ucsd.edu [Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610 (United States)

    2012-01-15

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores, which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA. - Highlights: > Examined four aromatic groups as a means to detect hydroxyl radicals by fluorescence. > Coumarin system suffers from the fewest disadvantages. > Characterized its reactivity when linked to a hexa-arginine peptide.

  1. Inhibitory effect of membrane-binding drugs on excision repair of DNA damage in UV-irradiated Escherichia coli

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    Todo, T.; Yonei, S. (Kyoto Univ. (Japan). Lab. of Radiation Biology)

    1983-04-01

    The effects of procaine and lidocaine on DNA-repair processes were investigated in UV-irradiated cells of E. coli with different DNA-repair capacities. The cells were irradiated with various doses of UV and then incubated at 37/sup 0/C in M9 buffer (liquid-holding) or in EM9 medium in the presence or absence of membrane-binding drugs. The results obtained are as follows. (1) In strains H/r30 (wild-type for DNA repair) and NG30 (recA/sup -/), the increase in survival with increase in time of liquid-holding was almost completely inhibited by the addition of procaine and lidocaine. The same trends were observable under conditions of post-irradiation incubation in EM9 medium, more efficiently in recA/sup -/ strain than in the wild-type strain. (2) The addition of these drugs gave an apparent enhancement of the frequency of UV-induced mutation to arginine prototrophy, corresponding to a decrease in survival. (3) There were negligible effects of the drugs on survival and mutation in the excision-repair-defective strain, Hs30 (uvrB/sup -/). (4) The removal of thymine dimers from DNA was actually reduced by the addition of procaine.

  2. CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

    Science.gov (United States)

    Ramirez-Garcés, Diana; Camborde, Laurent; Pel, Michiel J C; Jauneau, Alain; Martinez, Yves; Néant, Isabelle; Leclerc, Catherine; Moreau, Marc; Dumas, Bernard; Gaulin, Elodie

    2016-04-01

    To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR.

  3. The DNA damage-binding protein XPC is a frequent target for inactivation in squamous cell carcinomas.

    Science.gov (United States)

    de Feraudy, Sebastien; Ridd, Katie; Richards, Lauren M; Kwok, Pui-Yan; Revet, Ingrid; Oh, Dennis; Feeney, Luzviminda; Cleaver, James E

    2010-08-01

    XPC, the main damage-recognition protein responsible for nucleotide excision repair of UVB damage to DNA, is lost or mutated in xeroderma pigmentosum group C (XP-C), a rare inherited disease characterized by high incidence and early onset of non-melanoma and melanoma skin cancers. The high incidence of skin cancers in XP-C patients suggests that loss of expression of XPC protein might also provide a selective advantage for initiation and progression of similar cancers in non XP-C patients in the general population. To test whether XPC is selectively lost in squamous cell carcinomas from non XP-C patients, we examined XPC expression by immunohistochemistry on a tissue microarray with 244 tissue cores, including in situ and invasive squamous-cell carcinomas (SCCs), keratoacanthoma (KA), and normal skin samples from both immunocompetent and immunosuppressed patients. We found that XPC expression was lost in 49% of invasive squamous cell carcinomas from immunocompetent patients and 59% from immunosuppressed patients. Loss of expression was correlated with deletions of chromosomal 3p and mutations in the XPC gene. The XPC gene is consequently inactivated or lost in almost half of squamous cell carcinomas from non XP-C patients. Loss or mutation of XPC may be an early event during skin carcinogenesis that provides a selective advantage for initiation and progression of squamous cell carcinomas in non XP-C patients.

  4. The RNA Splicing Response to DNA Damage.

    Science.gov (United States)

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

  5. DNA damage in neurodegenerative diseases

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-15

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

  6. Mechanism of DNA damage tolerance

    Institute of Scientific and Technical Information of China (English)

    Xin; Bi

    2015-01-01

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

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

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

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

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

    Science.gov (United States)

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

    2014-03-01

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

  9. DNA binding studies of tartrazine food additive.

    Science.gov (United States)

    Kashanian, Soheila; Zeidali, Sahar Heidary

    2011-07-01

    The interaction of native calf thymus DNA with tartrazine in 10 mM Tris-HCl aqueous solution at neutral pH 7.4 was investigated. Tartrazine is a nitrous derivative and may cause allergic reactions, with a potential of toxicological risk. Also, tartrazine induces oxidative stress and DNA damage. Its DNA binding properties were studied by UV-vis and circular dichroism spectra, competitive binding with Hoechst 33258, and viscosity measurements. Tartrazine molecules bind to DNA via groove mode as illustrated by hyperchromism in the UV absorption band of tartrazine, decrease in Hoechst-DNA solution fluorescence, unchanged viscosity of DNA, and conformational changes such as conversion from B-like to C-like in the circular dichroism spectra of DNA. The binding constants (K(b)) of DNA with tartrazine were calculated at different temperatures. Enthalpy and entropy changes were calculated to be +37 and +213 kJ mol(-1), respectively, according to the Van't Hoff equation, which indicated that the reaction is predominantly entropically driven. Also, tartrazine does not cleave plasmid DNA. Tartrazine interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 3.75 × 10(4) M(-1).

  10. DNA damage in neurodegenerative diseases.

    Science.gov (United States)

    Coppedè, Fabio; Migliore, Lucia

    2015-06-01

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

  11. DNA damage and carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Stelow, R B

    1980-01-01

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

  12. Sperm DNA oxidative damage and DNA adducts

    Science.gov (United States)

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

    2015-01-01

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

  13. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

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

  14. Increased DNA binding activity of NF-κB, STAT-3, SMAD3 and AP-1 in acutely damaged liver

    Institute of Scientific and Technical Information of China (English)

    Adriana Salazar-Montes; Luis Ruiz-Corro; Ana SandovaI-Rodriguez; Alberto Lopez-Reyes; Juan Armendariz-Borunda

    2006-01-01

    AIM: To investigate the role of genes and kinetics of specific transcription factors in liver regeneration, and to analyze the gene expression and the activity of some molecules crucially involved in hepatic regeneration.METHODS: USING gel-shift assay and RT-PCR,transcription factors, such as NF-κB, STAT-3, SMAD3and AP-1, and gene expression of inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF) and c-met were analyzed in an animal model of chemically induced hepatectomy.RESULTS: Gene expression of HGF and its receptor c-met peaked at 3 h and 24 h after acute CCl4 intoxication. iNOS expression was only observed from 6 to 48 h.Transcriptional factor NF-κB had an early activation at 30min after acute liver damage. STAT-3 peaked 3 h postintoxication, while AP-1 displayed a peak of activation at 48 h. SMAD3 showed a high activity at all analyzed times.CONCLUSION: TNF-α and IL-6 play a central role in hepatic regeneration. These two molecules are responsible for triggering the cascade of events and switch-on of genes involved in cell proliferation, such as growth factors, kinases and cyclins which are direct participants of cell proliferation.

  15. High-Fidelity DNA Sensing by Protein Binding Fluctuations

    CERN Document Server

    Tlusty, Tsvi; Libchaber, Albert; 10.1103/PhysRevLett.93.258103

    2010-01-01

    One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response.We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

  16. The DNA damage response during mitosis

    NARCIS (Netherlands)

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

    2013-01-01

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

  17. The DNA damage response during mitosis

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-06-30

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

  19. Chromatin structure and DNA damage repair

    Directory of Open Access Journals (Sweden)

    Dinant Christoffel

    2008-11-01

    Full Text Available Abstract The integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. These damaging agents can induce a wide variety of lesions in the DNA, such as double strand breaks, single strand breaks, oxidative lesions and pyrimidine dimers. The cell has evolved intricate DNA damage response mechanisms to counteract the genotoxic effects of these lesions. The two main features of the DNA damage response mechanisms are cell-cycle checkpoint activation and, at the heart of the response, DNA repair. For both damage signalling and repair, chromatin remodelling is most likely a prerequisite. Here, we discuss current knowledge on chromatin remodelling with respect to the cellular response to DNA damage, with emphasis on the response to lesions resolved by nucleotide excision repair. We will discuss the role of histone modifications as well as their displacement or exchange in nucleotide excision repair and make a comparison with their requirement in transcription and double strand break repair.

  20. Using DNA damage to monitor water environment

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

  1. DNA damage in plant herbarium tissue.

    NARCIS (Netherlands)

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

    2011-01-01

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

  2. The helical structure of DNA facilitates binding

    Science.gov (United States)

    Berg, Otto G.; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

    2016-09-01

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  3. DNA Damage Signals and Space Radiation Risk

    Science.gov (United States)

    Cucinotta, Francis A.

    2011-01-01

    Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

  4. Cellular responses to environmental DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    1994-08-01

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

  5. DNA damage in plant herbarium tissue.

    Directory of Open Access Journals (Sweden)

    Martijn Staats

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

  6. DNA Damage in Plant Herbarium Tissue

    Science.gov (United States)

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

    2011-01-01

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

  7. Apoptosis and DNA damage in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    R John Aitken; Adam J Koppers

    2011-01-01

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

  8. SIRT participates at DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-15

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

  9. The DNA damage response in mammalian oocytes

    Directory of Open Access Journals (Sweden)

    John eCarroll

    2013-06-01

    Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

  10. The DNA damage response during mitosis.

    Science.gov (United States)

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

    2013-10-01

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

  11. The DNA damage response during mitosis

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-15

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

  12. Damage, DNA Repair, Aging, and Neurodegeneration

    Science.gov (United States)

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

    2017-01-01

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

  13. Tetrameric ZBRK1 DNA binding domain has affinity towards cognate DNA in absence of zinc ions.

    Science.gov (United States)

    Yadav, Lumbini R; Biswal, Mahamaya N; Vikrant; Hosur, M V; Varma, Ashok K

    2014-07-18

    Zinc finger transcription regulatory proteins play crucial roles in cell-cycle regulation, DNA damage response and tumor genesis. Human ZBRK1 is a zinc-finger transcription repressor protein, which recognizes double helical DNA containing consensus sequences of 5'GGGXXXCAGXXXTTT3'. In the present study, we have purified recombinant DNA binding domain of ZBRK1, and studied binding with zinc ions and DNA, using biophysical techniques. The elution profile of the purified protein suggests that this ZBRK1 forms a homotetramer in solution. Dissociation and pull down assays also suggest that this domain forms a higher order oligomer. The ZBRK1-DNA binding domain acquires higher stability in the presence of zinc ions and DNA. The secondary structure of the ZBRK1-DNA complex is found to be significantly altered from the standard B-DNA conformation. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Experimental study of oxidative DNA damage

    DEFF Research Database (Denmark)

    Loft, S; Deng, Xiaohong; Tuo, J

    1998-01-01

    compounds have been studied in animal experiments mainly in rats and mice, and generally with measurement of 8-oxodG with HPLC-EC. A large number of well-known carcinogens induce 8-oxodG formation in liver and/or kidneys. Moreover several animal studies have shown a close relationship between induction...... of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran......, potassium bromate and paraquat, are less potent and/or more organ specific. Inflammation and activation of an inflammatory response by phorbol esters or E. coli lipopolysaccharide (LPS) induce oxidative DNA damage in many target cells and enhance benzene-induced DNA damage in mouse bone marrow. Experimental...

  15. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-03-24

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

  16. A novel cis-acting element from the 3′UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression

    Science.gov (United States)

    Melanson, Brian D.; Cabrita, Miguel A.; Bose, Reetesh; Hamill, Jeffrey D.; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A.; Zhao, Tong T.; Holcik, Martin; McKay, Bruce C.

    2013-01-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3′ untranslated region (3′UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3′UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3′UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3′UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3′UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked. PMID:23605047

  17. A novel cis-acting element from the 3'UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression.

    Science.gov (United States)

    Melanson, Brian D; Cabrita, Miguel A; Bose, Reetesh; Hamill, Jeffrey D; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A; Zhao, Tong T; Holcik, Martin; McKay, Bruce C

    2013-06-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3' untranslated region (3'UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3'UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3'UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3'UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3'UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked.

  18. Polyomavirus interaction with the DNA damage response

    Institute of Scientific and Technical Information of China (English)

    Joshua; L.Justice; Brandy; Verhalen; Mengxi; Jiang

    2015-01-01

    Viruses are obligate intracellular parasites that subvert cellular metabolism and pathways to mediate their own replication—normally at the expense of the host cell. Polyomaviruses are a group of small DNA viruses, which have long been studied as a model for eukaryotic DNA replication. Polyomaviruses manipulate host replication proteins, as well as proteins involved in DNA maintenance and repair, to serve as essential cofactors for productive infection. Moreover, evidence suggests that polyomavirus infection poses a unique genotoxic threat to the host cell. In response to any source of DNA damage, cells must initiate an effective DNA damage response(DDR) to maintain genomic integrity, wherein two protein kinases, ataxia telangiectasia mutated(ATM) and ATM- and Rad3-related(ATR), are major regulators of DNA damage recognition and repair. Recent investigation suggests that these essential DDR proteins are required for productive polyomavirus infection. This review will focus on polyomaviruses and their interaction with ATMand ATR-mediated DNA damage responses and the effect of this interaction on host genomic stability.

  19. DNA damage response in adult stem cells.

    Science.gov (United States)

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

    2014-04-01

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

  20. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

    NARCIS (Netherlands)

    Luijsterburg, Martijn S.; Goedhart, Joachim; Moser, Jill; Kool, Hanneke; Geverts, Bart; Houtsmuller, Adriaan B.; Mullenders, Leon H. F.; Vermeulen, Wim; van Driel, Roel

    2007-01-01

    Damage DNA binding protein 2 ( DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and

  1. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC.

    NARCIS (Netherlands)

    Luijsterburg, M.S.; Goedhart, J.; Moser, J.; Kool, H.; Geverts, B.; Houtsmuller, A.B.; Mullenders, L.H.; Vermeulen, W.; van Driel, R.

    2007-01-01

    Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A

  2. Binding sequences for RdgB, a DNA damage-responsive transcriptional activator, and temperature-dependent expression of bacteriocin and pectin lyase genes in Pectobacterium carotovorum subsp. carotovorum.

    Science.gov (United States)

    Yamada, Kazuteru; Kaneko, Jun; Kamio, Yoshiyuki; Itoh, Yoshifumi

    2008-10-01

    Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23 degrees C) differs from that for synthesis of Pnl (30 degrees C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P(0), P(1), and P(2) promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K(d) [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K(d) = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23 degrees C compared with that at 30 degrees C. In contrast, the amount of pnl transcription tripled at 30 degrees C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30 degrees C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

  3. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    , 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......Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA...... degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes...

  4. Variables influencing DNA-binding in mouse liver.

    Science.gov (United States)

    Neumann, H G

    1987-01-01

    The suitability of certain mouse strains for carcinogenicity testing has been questioned. Some chemicals increase the incidence of liver tumors above a relatively high background, an effect not seen in rats. This raises the question whether species and tissue specific effects are involved which are reflected in the DNA binding of metabolites. DNA binding indices in mouse liver have been determined in only a few instances. They are comparable to those found for rat liver DNA with aniline, benzo(a)-pyrene, butadiene, dimethylnitrosamine, methylnitrosourea and they are lower in the mouse with aflatoxin B1, trans-4-acetylaminostilbene and 2-aminofluorene derivatives. The available data on DNA binding in mouse liver suggest that the same adducts are formed as in rats but that metabolism and repair are variables which can modify the extent of DNA damage. However, the extent of DNA binding does not always correlate with the susceptibility of this tissue to carcinogenesis. But mouse liver is no exception in this respect. It is concluded that the formation of mouse liver tumors in long term studies with genotoxic chemicals indicates tumor initiating potential. In contrast, there are other chemicals such as chlorinated hydrocarbon insecticides which do not bind to DNA to any extent and which are not genotoxic in common short term tests and yet give rise to liver tumors in mice but not in rats. Positive results in long term studies are suggested to indicate promoting properties of such compounds.

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Vitamin C for DNA damage prevention

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-01

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

  7. NAD+ Modulates p53 DNA Binding Specificity and Function

    Science.gov (United States)

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

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

    Science.gov (United States)

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

    2014-04-15

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

  9. Homologous recombination in DNA repair and DNA damage tolerance

    Institute of Scientific and Technical Information of China (English)

    Xuan Li; Wolf-Dietrich Heyer

    2008-01-01

    Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical sup-port for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modaUties of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

  10. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    Science.gov (United States)

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

  11. Structure-based analysis of HU-DNA binding.

    Science.gov (United States)

    Swinger, Kerren K; Rice, Phoebe A

    2007-01-26

    HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

  12. Structure-based Analysis to Hu-DNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Swinger,K.; Rice, P.

    2007-01-01

    HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from 10-14.5 kcal/mol, representing K{sub d} values in the nanomolar to low picomolar range, and a maximum stabilization of at least 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

  13. Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Roswell Park Memorial Institute, Buffalo, NY (USA))

    1987-06-01

    A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.

  14. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

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

  15. Involvement of 53BP1, a p53 Binding Protein, in Chk2 Phosphyorylation of p53 and DNA Damage Cell Cycle Checkpoints

    Science.gov (United States)

    2006-05-01

    20:4859-69. 24. Cote J, Utley RT, Workman JL. Basic analysis of transcription factor binding to nucleo - somes. Methods Mol Gen 1995; 6:108-27. 25...47755 (2001). 18. D. Cortez , Y. Wang, J. Qin, S. J. Elledge, Science 286, 1162 (1999). 19. R. DiTullio, T. Halazonetis, personal communication. 20. A...Cancer Res. 83, 209 (2001). 23. Antibodies to Chk2T68P provided by J. Chen; 53BP1, T. D. Halazonetis; and -H2AX, W. M. Bonner. 24. We thank D. Cortez

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

    Science.gov (United States)

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

    2017-02-27

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

  17. DNA damage checkpoint recovery and cancer development

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-10

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

  18. SUMO-1 possesses DNA binding activity

    Directory of Open Access Journals (Sweden)

    Wieruszeski Jean-Michel

    2010-05-01

    Full Text Available Abstract Background Conjugation of small ubiquitin-related modifiers (SUMOs is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs. Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood. Findings Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM. Conclusion This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

  19. Profiling oxidative DNA damage: effects of antioxidants.

    Science.gov (United States)

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

    2012-11-01

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

  20. An immunochemical assay to detect DNA damage in bovine sperm

    NARCIS (Netherlands)

    Schans, G.P. van der; Haring, R.; Dijk- Knijnenburg, H.C.M. van; Bruijnzeel, P.L.B.; Daas, N.H.G. den

    2000-01-01

    An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of

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

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

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

  2. Chromatin remodeling in the UV-induced DNA damage response

    NARCIS (Netherlands)

    Ö.Z. Aydin (Özge)

    2014-01-01

    markdownabstract__Abstract__ DNA damage interferes with transcription and replication, causing cell death, chromosomal aberrations or mutations, eventually leading to aging and tumorigenesis (Hoeijmakers, 2009). The integrity of DNA is protected by a network of DNA repair and associated signalling

  3. Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

    Directory of Open Access Journals (Sweden)

    Biswas-Fiss Esther E

    2012-06-01

    Full Text Available Abstract Background Single-stranded DNA binding proteins (SSB are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase. Results We have cloned and purified SSB from Bacillus anthracis (SSBBA. In the absence of DNA, at concentrations ≤100 μg/ml, SSBBA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSBBA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT70 binding suggested that SSBBA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure. Conclusions Unlike other prokaryotic SSB proteins, SSBBA from Bacillus anthracis appeared to be monomeric at concentrations ≤100 μg/ml as determined by SE-HPLC. SSBBA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSBBA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSBBA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.

  4. ATM and ATR:Sensing DNA damage

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Zheng-Ping Xu; Yun Huang; Hope E. Hamrick; Penelope J. Duerksen-Hughes; Ying-Nian Yu

    2004-01-01

    Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade. Although the initial activation of protein kinase cascade is not fully understood,human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progresses in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of genotoxic stress response.

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

    Science.gov (United States)

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

    2006-10-01

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

  6. Protection of DNA damage by radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

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

    1998-12-01

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

  7. Ion irradiation induced direct damage to DNA

    CERN Document Server

    Wang, Wei; Su, Wenhui

    2008-01-01

    Ion beams have been widely applied in a few biological research fields such as radioactive breeding, health protection, and tumor therapy. Up to now many interesting and impressive achievements in biology and agriculture have been made. Over the past several decades, scientists in biology, physics, and chemistry have pursued investigations focused on understanding the mechanisms of these radiobiological effects of ion beams. From the chemical point of view, these effects are due to the ion irradiation induced biomolecular damage, direct or indirect. In this review, we will present a chemical overview of the direct effects of ion irradiation upon DNA and its components, based on a review of literature combined with recent experimental results. It is suggested that, under ion bombardment, a DNA molecule undergoes a variety of processes, including radical formation, atomic displacement, intramolecular bond-scissions, emission of fragments, fragment recombination and molecular crosslink, which may lead to genetic...

  8. Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

    Science.gov (United States)

    Pietrzak, Monika; Halicka, H Dorota; Wieczorek, Zbigniew; Wieczorek, Jolanta; Darzynkiewicz, Zbigniew

    2008-06-01

    We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

  9. On binding specificity of (6–4) photolyase to a T(6–4)T DNA photoproduct

    DEFF Research Database (Denmark)

    Aalbæk Jepsen, Katrine; Solov'yov, Ilia

    2017-01-01

    this binding for a specific enzyme called (6–4) photolyase, which is capable of repairing certain UV-induced damage in DNA. Through molecular dynamics simulations we describe the binding between photolyase and the DNA and reveal that several charged amino acid residues in the enzyme, such as arginines...... and lysines turn out to be important. Especially R421 is crucial, as it keeps the DNA strands at the damaged site inside the repair pocket of the enzyme separated. DNA photolyase is structurally highly homologous to a protein called cryptochrome. Both proteins are biologically activated similarly, namely......Abstract: Different factors lead to DNA damage and if it is not repaired in due time, the damaged DNA could initiate mutagenesis and cancer. To avoid this deadly scenario, specific enzymes can scavenge and repair the DNA, but the enzymes have to bind first to the damaged sites. We have investigated...

  10. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways

    OpenAIRE

    Ichijima, Yosuke; Sin, Ho-Su; Satoshi H Namekawa

    2012-01-01

    Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR p...

  11. 14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

    OpenAIRE

    Mohammad, Duaa H.; Yaffe, Michael B.

    2009-01-01

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as p...

  12. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  13. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Michael Fasullo

    2015-04-01

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

  14. Regulation of HuR by DNA Damage Response Kinases

    Directory of Open Access Journals (Sweden)

    Hyeon Ho Kim

    2010-01-01

    Full Text Available As many DNA-damaging conditions repress transcription, posttranscriptional processes critically influence gene expression during the genotoxic stress response. The RNA-binding protein HuR robustly influences gene expression following DNA damage. HuR function is controlled in two principal ways: (1 by mobilizing HuR from the nucleus to the cytoplasm, where it modulates the stability and translation of target mRNAs and (2 by altering its association with target mRNAs. Here, we review evidence that two main effectors of ataxia-telangiectasia-mutated/ATM- and Rad3-related (ATM/ATR, the checkpoint kinases Chk1 and Chk2, jointly influence HuR function. Chk1 affects HuR localization by phosphorylating (hence inactivating Cdk1, a kinase that phosphorylates HuR and thereby blocks HuR's cytoplasmic export. Chk2 modulates HuR binding to target mRNAs by phosphorylating HuR's RNA-recognition motifs (RRM1 and RRM2. We discuss how HuR phosphorylation by kinases including Chk1/Cdk1 and Chk2 impacts upon gene expression patterns, cell proliferation, and survival following genotoxic injury.

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

    Directory of Open Access Journals (Sweden)

    Björn Schumacher

    2016-08-01

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

  16. Studies on DNA Damage Response in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Han, Wenyuan

    global reactions known as DNA damage response (DDR). In Bacteria and Eukaryotes, the global reactions include a series of transcription regulations and protein post-translation modifications, which can activate DNA repair machineries, suppress cell division and delay DNA replication, and induce......All living organisms have to keep their genetic information intact. However, environmental stimuli and endogenous factors constantly yield various DNA lesions, which impose serious challenges for cells to maintain the stability of their genetic materials. Upon severe DNA damage, cells initiate...... programmed cell death (PCD) upon lethal DNA damage. However, little is known about DNA damage response in Archaea. To start to address the problem, I investigated the general cellular response of Sulfolobus islandicus, a model organism of Archaea, to DNA damage agents, 4-nitroquinoline 1-oxide (NQO...

  17. DBD2BS: connecting a DNA-binding protein with its binding sites

    OpenAIRE

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes c...

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

    Science.gov (United States)

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

    2017-01-01

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

  19. Cytotoxicity and DNA damage associated with pyrazoloacridine in MCF-7 breast cancer cells.

    Science.gov (United States)

    Grem, J L; Politi, P M; Berg, S L; Benchekroun, N M; Patel, M; Balis, F M; Sinha, B K; Dahut, W; Allegra, C J

    1996-06-28

    We examined the effects of pyrazoloacridine (PZA), an investigational anticancer agent in clinical trials, on cytotoxicity, DNA synthesis, and DNA damage in MCF-7 human breast carcinoma cells. With PZA concentrations ranging from 0.5 to 50 microM for durations of 3-72 hr, cytotoxicity increased in proportion to the total PZA exposure (concentration x time). Inhibition of DNA and RNA syntheses increased with increasing PZA concentration x time (microM.hr). A 24-hr exposure to 1 and 10 microM PZA reduced DNA synthesis to 62 and 5% of control, respectively, decreased the proportion of cells in S phase with accumulation of cells in G2 + M phase, and inhibited cell growth at 72 hr by 68 and 100%. Newly synthesized DNA was more susceptible to damage during PZA exposure, with subsequent induction of parental DNA damage. Significant damage to newly synthesized DNA as monitored by alkaline elution was evident after a 3-hr exposure to > or = 5 microM PZA. Longer PZA exposures (> or = 10 microM for 16 hr) were required to elicit damage to parental DNA. Induction of single-strand breaks in parental DNA correlated closely with induction of double-strand breaks and detachment of cells from the monolayer. PZA-mediated DNA fragmentation was not accompanied by the generation of oligonucleosomal laddering in MCF-7 cells, but induction of very high molecular weight DNA fragmentation (0.5 to 1 Mb) was detected by pulsed-field gel electrophoresis. In vitro binding of PZA to linear duplex DNA (1 kb DNA ladder) and closed, circular plasmid DNA was demonstrated by a shift in migration during agarose electrophoresis. PZA interfered with topoisomerase I- and II-mediated relaxation of plasmid DNA in a cell-free system, but the cytotoxic effects of PZA did not appear to involve a direct interaction with topoisomerase I or II (stabilization of the topoisomerase I- or II-DNA cleavable complex). PZA-mediated cytotoxicity correlated strongly with inhibition of DNA and RNA syntheses, and damage to

  20. Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity.

    Directory of Open Access Journals (Sweden)

    Shu-Huei Wang

    Full Text Available Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity.

  1. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  2. Thermodynamics of the DNA damage repair steps of human 8-oxoguanine DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Nikita A Kuznetsov

    Full Text Available Human 8-oxoguanine DNA glycosylase (hOGG1 is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG. In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van't Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.

  3. Endothelial IL-33 Expression Is Augmented by Adenoviral Activation of the DNA Damage Machinery.

    Science.gov (United States)

    Stav-Noraas, Tor Espen; Edelmann, Reidunn J; Poulsen, Lars La Cour; Sundnes, Olav; Phung, Danh; Küchler, Axel M; Müller, Fredrik; Kamen, Amine A; Haraldsen, Guttorm; Kaarbø, Mari; Hol, Johanna

    2017-04-15

    IL-33, required for viral clearance by cytotoxic T cells, is generally expressed in vascular endothelial cells in healthy human tissues. We discovered that endothelial IL-33 expression was stimulated as a response to adenoviral transduction. This response was dependent on MRE11, a sensor of DNA damage that can also be activated by adenoviral DNA, and on IRF1, a transcriptional regulator of cellular responses to viral invasion and DNA damage. Accordingly, we observed that endothelial cells responded to adenoviral DNA by phosphorylation of ATM and CHK2 and that depletion or inhibition of MRE11, but not depletion of ATM, abrogated IL-33 stimulation. In conclusion, we show that adenoviral transduction stimulates IL-33 expression in endothelial cells in a manner that is dependent on the DNA-binding protein MRE11 and the antiviral factor IRF1 but not on downstream DNA damage response signaling. Copyright © 2017 by The American Association of Immunologists, Inc.

  4. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.

    Science.gov (United States)

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2013-07-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.

  5. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  6. DNA-damage response control of E2F7 and E2F8

    OpenAIRE

    Panagiotis Zalmas, L.; Zhao, Xiujie; Graham, Anne L; FISHER Rebecca; Reilly, Carmel; Coutts, Amanda S; La Thangue, Nicholas B

    2008-01-01

    Here, we report that the two recently identified E2F subunits, E2F7 and E2F8, are induced in cells treated with DNA-damaging agents where they have an important role in dictating the outcome of the DNA-damage response. The DNA-damage-dependent induction coincides with the binding of E2F7 and E2F8 to the promoters of certain E2F-responsive genes, most notably that of the E2F1 gene, in which E2F7 and E2F8 coexist in a DNA-binding complex. As a consequence, E2F7 and E2F8 repress E2F target genes...

  7. Aging and oxidatively damaged nuclear DNA in animal organs

    DEFF Research Database (Denmark)

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

    2010-01-01

    Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs...... with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies...... evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation....

  8. Single Molecule Scanning of DNA Radiation Oxidative Damage Project

    Data.gov (United States)

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

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

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

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

  10. Characterization of the DNA binding properties of polyomavirus capsid protein

    Science.gov (United States)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  11. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    OpenAIRE

    Joerg Kotzerke; Robert Punzet; Roswitha Runge; Sandra Ferl; Liane Oehme; Gerd Wunderlich; Robert Freudenberg

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments ca...

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

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    Organisms release DNA both when they live and die. Eventually the DNA disintegrates entirely or it is re-metabolized. There is a constant deposition and decomposition that maintains an environmental pool with large quantities of extracellular DNA, some of which can be thousands of years old...... it by damaged short DNA with abasic sites, crosslinks, and miscoding lesions, which are the most common damages in environmental DNA. This is emphasized by successful natural transformation by 43,000-year-old DNA. We find that the process is a simple variant of natural transformation. On top, we illustrate...... acquire functional genetic signatures of the deeper past. Moreover, not only can old DNA revert microbes to past genotypes, but damaged DNA can also produce new variants of already functional sequences. Besides, DNA fragments carry potential to combine functional domains in new ways. The identified novel...

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

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    it by damaged short DNA with abasic sites, crosslinks, and miscoding lesions, which are the most common damages in environmental DNA. This is emphasized by successful natural transformation by 43,000-year-old DNA. We find that the process is a simple variant of natural transformation. On top, we illustrate......Organisms release DNA both when they live and die. Eventually the DNA disintegrates entirely or it is re-metabolized. There is a constant deposition and decomposition that maintains an environmental pool with large quantities of extracellular DNA, some of which can be thousands of years old....... The degrading DNA is fragmented and damaged, often to less than one hundred base pairs. Such DNA is only recognized as microbial nutrients and is not considered as direct contributors to bacterial evolutionary processes. The main study shows natural transformation by very short DNA (≥20bp). Further we also show...

  14. Cancer risk and oxidative DNA damage in man

    DEFF Research Database (Denmark)

    Loft, Steffen; Poulsen, H E

    1996-01-01

    of Brussels sprouts reduced the oxidative DNA damage rate, estimated by the urinary excretion of 8-oxodG, and the intake of vitamin C was a determinant for the level of 8-oxodG in sperm DNA. A low-fat diet reduced another marker of oxidative DNA damage in leukocytes. In patients with diseases associated...... with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion...... of oxidative DNA damage as an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still lacking. This could possibly be supported by demonstration of the rate of oxidative DNA damage as an independent risk factor for cancer in a prospective study...

  15. Binding characteristics of salbutamol with DNA by spectral methods

    Science.gov (United States)

    Bi, Shuyun; Pang, Bo; Zhao, Tingting; Wang, Tianjiao; Wang, Yu; Yan, Lili

    2013-07-01

    Salbutamol interacting with deoxyribonucleic acid (DNA) was examined by fluorescence, UV absorption, viscosity measurements, and DNA melting techniques. The binding constants and binding sites were obtained at different temperatures by fluorescence quenching. The Stern-Volmer plots showed that the quenching of fluorescence of salbutamol by DNA was a static quenching. To probe the binding mode, various analytical methods were performed and the results were as follows: hyperchromic effect was shown in the absorption spectra of salbutamol upon addition of DNA; there was no appreciable increase in melting temperature of DNA when salbutamol was presented in DNA solution; the fluorescence intensity of salbutamol-DNA decrease with the increasing ionic strength; the relative viscosity of DNA did not change in the presence of salbutamol; the binding constant of salbutamol with double strand DNA (dsDNA) was much higher than that of it with single strand DNA (ssDNA). All these results indicated that the binding mode of salbutamol to DNA should be groove binding. The thermodynamic parameters suggested that hydrogen bond or van der Waals force might play an important role in salbutamol binding to DNA. According to the Förster energy transference theory, the binding distance between the acceptor and donor was 3.70 nm.

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

    Science.gov (United States)

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

    2012-07-06

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

  17. Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

    Science.gov (United States)

    Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

    2015-05-12

    VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

  18. Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

    Science.gov (United States)

    Chen, Pin-I; Cao, Aiqin; Miyagawa, Kazuya; Tojais, Nancy F.; Hennigs, Jan K.; Li, Caiyun G.; Sweeney, Nathaly M.; Inglis, Audrey S.; Wang, Lingli; Li, Dan; Ye, Matthew; Feldman, Brian J.

    2017-01-01

    Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress. PMID:28138562

  19. A Microscopic Study of the DNA Damage Response

    NARCIS (Netherlands)

    C. Dinant (Christoffel)

    2008-01-01

    textabstractThe integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. These damaging agents can induce a wide variety of lesions in the DNA, such as double strand breaks (DSB), single strand breaks (SSB), oxidative lesions and pyrimidine dimers. The

  20. Delineating the DNA damage response using systems biology approaches

    NARCIS (Netherlands)

    Stechow, Louise von

    2013-01-01

    Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted

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

    Science.gov (United States)

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

    2013-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response. PMID:23346550

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

    Institute of Scientific and Technical Information of China (English)

    Rong Tan; Li Lan

    2016-01-01

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

  3. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

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

  4. Cancer risk and oxidative DNA damage in man

    DEFF Research Database (Denmark)

    Loft, S; Poulsen, H E

    1996-01-01

    of damage and the balance between the damage and repair rate, respectively. By means of biomarkers a number of important factors have been studied in humans. Ionizing radiation, a carcinogenic and pure source of ROS, induced both urinary and leukocyte biomarkers of oxidative DNA damage. Tobacco smoking......, another carcinogenic source of ROS, increased the oxidative DNA damage rate by 35-50% estimated from the urinary excretion of 8-oxodG, and the level of 8-oxodG in leukocytes by 20-50%. The main endogenous source of ROS, the oxygen consumption, showed a close correlation with the 8-oxodG excretion rate...... of oxidative DNA damage as an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still lacking. This could possibly be supported by demonstration of the rate of oxidative DNA damage as an independent risk factor for cancer in a prospective study...

  5. DNA damage response and Autophagy: a meaningful partnership

    Directory of Open Access Journals (Sweden)

    ARISTIDES G ELIOPOULOS

    2016-11-01

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

  6. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.

    Science.gov (United States)

    Kolas, Nadine K; Chapman, J Ross; Nakada, Shinichiro; Ylanko, Jarkko; Chahwan, Richard; Sweeney, Frédéric D; Panier, Stephanie; Mendez, Megan; Wildenhain, Jan; Thomson, Timothy M; Pelletier, Laurence; Jackson, Stephen P; Durocher, Daniel

    2007-12-07

    Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.

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

    Science.gov (United States)

    Polo, Sophie E

    2015-02-13

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

  8. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

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

  9. Distribution patterns of postmortem damage in human mitochondrial DNA

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Willerslev, Eske; Hansen, Anders J

    2002-01-01

    The distribution of postmortem damage in mitochondrial DNA retrieved from 37 ancient human DNA samples was analyzed by cloning and was compared with a selection of published animal data. A relative rate of damage (rho(v)) was calculated for nucleotide positions within the human hypervariable region......, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed "low-diversity 1," through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions...

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

    Science.gov (United States)

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

    2015-01-01

    A variety of environmental stresses like chemicals, UV and ionizing radiation and organism's endogenous processes such as replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damage plays a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g., X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e., single, complex DNA lesions etc. that can be used as DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data. PMID:26082923

  11. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple

  12. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  13. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  14. Experimental Investigation of DNA Damage Induced by Heavy Ions

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    DNA is considered the critical target for radiobiological effects. It is highly important to study DNAdamage induced by ionizing radiation. Especially DNA double strand breaks have been identified as themost initial damage. In this experiment, DNA double strand breaks induced by heavy ions wereinvestigated with atomic force microscopy (AFM).

  15. Cancer risk and oxidative DNA damage in man

    DEFF Research Database (Denmark)

    Loft, Steffen; Poulsen, H E

    1996-01-01

    per 10(5) intact nucleosides. The damaged nucleosides accumulate with age in both nuclear and mitochondrial DNA. The products of repair of these lesions are excreted into the urine in amounts corresponding to a damage rate of up to 10(4) modifications in each cell every day. The most abundant...... with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion...... of oxidative DNA damage as an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still lacking. This could possibly be supported by demonstration of the rate of oxidative DNA damage as an independent risk factor for cancer in a prospective study...

  16. MicroRNAs: new players in the DNA damage response

    Institute of Scientific and Technical Information of China (English)

    Hailiang Hu; Richard A. Gatti

    2011-01-01

    The DNA damage response (DDR) is a signal transduction pathway that decides the cell's fate either to repair DNA damage or to undergo apoptosis if there is too much damage. Post-translational modifications modulate the assembly and activity of protein complexes during the DDR pathways. MicroRNAs (miRNAs) are emerging as a class of endogenous gene modulators that control protein levels, thereby adding a new layer of regulation to the DDR. In this review, we describe a new role for miRNAs in regulating the cellular response to DNA damage with a focus on DNA double-strand break damage. We also discuss the implications of miRNA's role in the DDR to stem cells, including embryonic stem cells and cancer stem cells, stressing the potential applications for miRNAs to be used as sensitizers for cancer radiotherapy and chemotherapy.

  17. Cooperative binding interaction of ethidium with allosteric DNA.

    Science.gov (United States)

    Suh, D

    1999-09-30

    The specific association of drugs with deoxyoligonucleotides, containing a B-Z junction between left-handed Z-DNA and right-handed B-DNA, was examined by fluorescence and circular dichroism (CD) technique. Ethidium was chosen for a simple DNA binding compound because it binds to right-handed DNA and hybrid B-Z forms containing a B-Z junction in a highly cooperative manner. The binding isotherms were analyzed by an allosteric model in order to describe the cooperativity of association. Binding of ethidium to the DNA that are initially in the hybrid B-Z forms showed over an order of magnitude higher affinity than other DNA which were entirely in the B-form. The conformational transitions of deoxyoligonucleotides containing a B-Z junction as a result of ethidium binding were monitored by CD and the influence of NaCl on the complex formation was also determined by the CD spectra. The singular value decomposition (SVD) analysis was used to characterize a family of CD spectra of the species in binding equilibria. The results of SVD analysis showed a strikingly complex thermodynamic equilibria of cooperative binding of drugs to the allosterically converted DNA forms. The results also showed that these DNA forms in low- and high-salt were different in the absence or presence of drug. These results demonstrate that DNA-binding-drugs can preferentially interact with specific DNA structures and that these interactions are accompanied by allosteric changes of DNA conformations.

  18. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces cell cycle synchronization in different human osteosarcoma cell lines. The UV pulse also has a destabilizing...

  19. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect...

  20. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Joshua W Modell

    2014-10-01

    Full Text Available Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.

  1. A DNA damage-induced, SOS-independent checkpoint regulates cell division in Caulobacter crescentus.

    Science.gov (United States)

    Modell, Joshua W; Kambara, Tracy K; Perchuk, Barrett S; Laub, Michael T

    2014-10-01

    Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.

  2. A DNA Damage-Induced, SOS-Independent Checkpoint Regulates Cell Division in Caulobacter crescentus

    Science.gov (United States)

    Modell, Joshua W.; Kambara, Tracy K.; Perchuk, Barrett S.; Laub, Michael T.

    2014-01-01

    Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage. PMID:25350732

  3. Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

    Science.gov (United States)

    Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

    2017-03-15

    NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability.

  4. Posttranscriptional regulation of gene expression – adding another layer of complexity to the DNA damage response

    Directory of Open Access Journals (Sweden)

    Jorge eBoucas

    2012-08-01

    Full Text Available In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR, is primarily thought to consist of two components – a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RNA-binding proteins and the control of their function through DNA damage-activated protein kinases.

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

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

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

  6. Imaging the DNA damage response with PET and SPECT

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-15

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

  7. Chromatin Modifications and the DNA Damage Response to Ionizing Radiation

    Directory of Open Access Journals (Sweden)

    Tej K Pandita

    2013-01-01

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

  8. AFM studies of nonspecific binding of enzyme on DNA

    Institute of Scientific and Technical Information of China (English)

    张益; 谢恒月; 等

    1996-01-01

    Atomic force microscope(AFM) is used to study restriction endonuclease digestion of plasmid DNA,pWRr plasmid DNA is digested by Hind Ⅲ,and the specific and the nonspecific binding of the restriction endonuclease are imaged,and the biological function of the enzyme binding to nonspecific sites is discussed.In addition,it is found that nonspecific binding of Hind ǚ could not induce the DNA characteristic bending angle.

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

    Science.gov (United States)

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

    2015-08-06

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

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

    Science.gov (United States)

    Demple, B; Halbrook, J

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

  11. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

  12. Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III*

    OpenAIRE

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Tomkinson, Alan E.; Ellenberger, Tom

    2008-01-01

    Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bin...

  13. Affinity purification of sequence-specific DNA binding proteins.

    OpenAIRE

    1986-01-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed t...

  14. Proteins in the nutrient-sensing and DNA damage checkpoint pathways cooperate to restrain mitotic progression following DNA damage.

    Directory of Open Access Journals (Sweden)

    Jennifer S Searle

    2011-07-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  16. Global chromatin fibre compaction in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Charlotte [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Hayward, Richard L. [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Gilbert, Nick, E-mail: Nick.Gilbert@ed.ac.uk [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom)

    2011-11-04

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

  17. Biomarkers of oxidative damage to DNA and repair

    DEFF Research Database (Denmark)

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

    2008-01-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation...... DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many...

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

    Directory of Open Access Journals (Sweden)

    Nidhi Nair

    2017-07-01

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

  19. BACH2: A marker of DNA damage and ageing

    NARCIS (Netherlands)

    L.M. Uittenboogaard (Lieneke); C. Payan-Gomez; J. Pothof (Joris); W.F.J. van IJcken (Wilfred); P.G. Mastroberardino (Pier); I. van der Pluijm (Ingrid); J.H.J. Hoeijmakers (Jan); M. Tresini (Maria)

    2013-01-01

    textabstractDNA damage and ageing share expression changes involving alterations in many aspects of metabolism, suppression of growth and upregulation of defence and genome maintenance systems. "Omics" technologies have permitted large-scale parallel measurements covering global cellular

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

    Science.gov (United States)

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

    2017-05-01

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

  1. Endogenous melatonin and oxidatively damaged guanine in DNA

    DEFF Research Database (Denmark)

    Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan

    2009-01-01

    attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. METHODS: Mother......-father-daughter(s) families (n = 55) were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr) has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua) results from the repair of DNA...... of oxidatively damaged guanine in DNA, thereby possibly increasing the risk of developing cancer. The possible different effects of melatonin in the rates of utilization of pathways for repair of oxidatively damaged guanine in DNA identified between older women and older men are intriguing....

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

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

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

  3. Typical Cell Signaling Response to Ionizing Radiation:DNA Damage and Extranuclear Damage

    Institute of Scientific and Technical Information of China (English)

    Hui Yu

    2012-01-01

    To treat many types of cancer,ionizing radiation (IR) is primarily used as external-beam radiotherapy,brachytherapy,and targeted radionuclide therapy.Exposure of tumor cells to IR can induce DNA damage as well as generation of reactiveoxygen species (ROS) and reactive nitrogen species (RNS) which can cause non-DNA lesions or extracellular damage like lipid perioxidation.The initial radiation-induced cell responses to DNA damage and ROS like the proteolytic processing,as well as synthesis and releasing ligands (such as growth factors,cytokines,and hormone) can cause the delayed secondary responses in irradiated and unirradiated bystander cells through paracrine and autocrine pathways.

  4. Synthesis, characterization, DNA binding, DNA cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

    Science.gov (United States)

    Thota, Sreekanth; Vallala, Srujana; Yerra, Rajeshwar; Rodrigues, Daniel Alencar; Raghavendra, Nulgumnalli Manjunathaiah; Barreiro, Eliezer J

    2016-01-01

    We report on the synthesis of novel Ru(II) compounds (Ru-1 to Ru-8) bearing R-pdc, 4-Cl-pbinh ligands (where R=4-CF3, 4-F, 4-OH pdc=3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, pbinh=phenoxybenzylidene isonicotinyl hydrazides) and their in vitro antitumor activity toward the cell lines murine leukemia L1210, human lymphocyte CEM, human epithelial cervical carcinoma HeLa, BEL-7402 and Molt4/C8. Some of the complexes exhibited more potent antiproliferative activity against cell lines than the standard drug cisplatin. Ruthenium complex Ru-2 displayed potent cytotoxicity with than that of cisplatin. DNA-binding, DNA cleavage and protein binding properties of ruthenium complexes with these ligands are reported. Interactions of these ruthenium complexes with DNA revealed an intercalative mode of binding between them. Synchronous fluorescence spectra proved that the interaction of ruthenium complexes with bovine serum albumin (BSA) resulted in a conformational change of the latter.

  5. The regulation of DNA damage tolerance by Ubiquitin and Ubiquitin-like modifiers

    Directory of Open Access Journals (Sweden)

    Lina Cipolla

    2016-06-01

    Full Text Available DNA replication is an extremely complex process that needs to be executed in a highly accurate manner in order to propagate the genome. This task requires the coordination of a number of enzymatic activities and it is incredibly fragile and prone to arrest after DNA damage. DNA damage tolerance provides a last line of defense that allows completion of DNA replication in presence of an unrepaired template. One of such mechanisms is called Post Replication Repair (PRR and it is used by the cells to bypass highly distorted templates caused by damaged bases. PRR is extremely important for the cellular life and performs the bypass of the damage both in an error free and in an error prone manner. In light of these two possible outcomes, PRR needs to be tightly regulated and controlled in order to prevent accumulation of mutations leading ultimately to genome instability. Post-translational modifications provide the framework for this regulation and Ubiquitylation and SUMOylation of PRR proteins play a pivotal role in choosing which pathway to activate, controlling the different outcomes of damage bypass. PCNA (Proliferating Cell Nuclear Antigen, the DNA clamp for replicative polymerases, plays a central role in the regulation of damage tolerance and its modification by Ubiquitin and SUMO controls both the error free and error prone branches of PRR. Furthermore, a significant number of polymerases involved in the bypass of DNA damage possess domains that can bind post-translational modifications and they are themselves target for ubiquitylation. In this review, we will focus on how Ubiquitin and Ubiquitin-like modifications can regulate the DNA damage tolerance systems and how they are capable of controlling the recruitment of different proteins to the replication fork.

  6. Radiation damage to DNA: the effect of LET

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-03-01

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

  7. Controlling the response to DNA damage by the APC/C-Cdh1.

    Science.gov (United States)

    de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

    2016-03-01

    Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

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

    Science.gov (United States)

    Hu, Jinchuan; Adar, Sheera

    2017-01-01

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

  9. Oxidative Damage to DNA and Its Relationship With Diabetic Complications

    Institute of Scientific and Technical Information of China (English)

    HONG-ZHI PAN; DONG CHANG; LEI-GUANG FENG; FENG-JUAN XU; HONG-YU KUANG; MING-JUN LU

    2007-01-01

    Objective To detect the oxidative DNA damage in diabetic patients and to investigate the relationship of oxidative DNA damage with diabetes and diabetic nephropathy. Methods Single cell gel electrophoresis (SCGE) was used to detect the DNA strand breaks in peripheral blood lymphocytes, and oxidative DNA damage product and serum 8-OHdG were determined by a competitive ELISA in 47 cases, including 25 patients without diabetic complications, 22 patients with diabetic nephropathy and 25 normal control subjects. Results Diabetic patients showed greater oxidative damage to DNA. The percentage of comet cells and the length of DNA migration (comet tail length) of peripheral blood lymphocytes were significantly increased in patients with diabetes, and significantly higher in patients with diabetic nephropathy than in diabetic patients without vascular complications (P<0.05). There was a significant increase in serum 8-OHdG in diabetic patients compared with normal subjects (P<0.05). Moreover, serum 8-OHdG was much higher in patients with diabetic nephropathy than in diabetic patients without vascular complications (P<0.05). Conclusion There is severe oxidative DNA damage in diabetic patients. Enhanced oxidative stress may be associated with diabetes, especially in patients with diabetic nephropathy.

  10. Can cytotoxic activity of anthracyclines be related to DNA damage?

    Science.gov (United States)

    Nishiyama, M; Horichi, N; Mazouzi, Z; Bungo, M; Saijo, N; Tapiero, H

    1990-02-01

    Accumulation, cytotoxicity, and DNA damages produced by doxorubicin (DOX), pirarubicin (THP-DOX), fluoro-doxorubicin (ME2303) or its isolated metabolite M1 have been investigated in human myelogenous leukemia cells, sensitive (K562) and resistant to DOX (K562/DOX). These compounds differed by lipophilicity and/or sugar moiety either with (DOX, THP-DOX) or without (ME2303, M1) amino group. In K562 cells, the cytotoxicity was correlated to DNA single-stranded breaks and the intracellular drug amount of DOX or M1. This was not true when the cells were treated with THP-DOX or ME2303. In addition, THP-DOX produced total DNA protein cross-linking. In K562 cells DNA damage was not repaired, while in K562/DOX repair of DNA damage produced by all drugs could be observed. Although in K562/DOX cells drug accumulation was much reduced, higher intracellular drug concentration was required to induce similar level of cytotoxicity and DNA damage. Thus, cytotoxicity produced by anthracycline is not always associated with DNA damage. Different level of resistance to DOX, THP-DOX, ME2303 or M1 is associated with reduced drug accumulation which varies with the structure.

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

    Science.gov (United States)

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

    2016-02-07

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

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

    Directory of Open Access Journals (Sweden)

    Xiaoguang Zhou

    2014-12-01

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

  13. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    Science.gov (United States)

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

  14. In vitro DNA binding studies of Aspartame, an artificial sweetener.

    Science.gov (United States)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

    2013-03-05

    A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1).

  15. Oxidatively damaged DNA in animals exposed to particles

    DEFF Research Database (Denmark)

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

    2013-01-01

    Exposure to combustion-derived particles, quartz and asbestos is associated with increased levels of oxidized and mutagenic DNA lesions. The aim of this survey was to critically assess the measurements of oxidatively damaged DNA as marker of particle-induced genotoxicity in animal tissues...

  16. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  17. Modulation of DNA binding by gene-specific transcription factors.

    Science.gov (United States)

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  18. Binding Parameters of Alkaloids Berberine and Sanguinarine with DNA

    CERN Document Server

    Gumenyuk, V G; Kutovyy, S Yu; Yashchuk, V M; Zaika, L A

    2012-01-01

    We study the interaction of berberine and sanguinarine (plant alkaloids) with DNA in aqueous solutions, by using optical spectroscopy methods (absorption and fluorescence). The dependencies of alkaloid spectral characteristics on the concentration ratio N/c between the DNA base pairs and alkaloid molecules in the solutions are considered, and the manifestations of the alkaloid-DNA binding are revealed. The character of binding is found to depend on N/c. The parameters of the binding of berberine and sanguinarine with DNA are determined, by using the modified Scatchard and McGhee-von Hippel equations

  19. Structure and mechanism of the UvrA-UvrB DNA damage sensor

    Energy Technology Data Exchange (ETDEWEB)

    Pakotiprapha, Danaya; Samuels, Martin; Shen, Koning; Hu, Johnny H; Jeruzalmi, David [Harvard

    2012-04-17

    Nucleotide excision repair (NER) is used by all organisms to eliminate DNA lesions. We determined the structure of the Geobacillus stearothermophilus UvrA-UvrB complex, the damage-sensor in bacterial NER and a new structure of UvrA. We observe that the DNA binding surface of UvrA, previously found in an open shape that binds damaged DNA, also exists in a closed groove shape compatible with native DNA only. The sensor contains two UvrB molecules that flank the UvrA dimer along the predicted path for DNA, ~80 Å from the lesion. We show that the conserved signature domain II of UvrA mediates a nexus of contacts among UvrA, UvrB and DNA. Further, in our new structure of UvrA, this domain adopts an altered conformation while an adjacent nucleotide binding site is vacant. Our findings raise unanticipated questions about NER and also suggest a revised picture of its early stages.

  20. Profiling DNA damage response following mitotic perturbations

    DEFF Research Database (Denmark)

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

    2016-01-01

    Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show...... phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude...

  1. Binding and interaction of di- and tri-substituted organometallic triptycene palladium complexes with DNA.

    Science.gov (United States)

    Kumari, Rina; Bhowmick, Sourav; Das, Neeladri; Das, Prolay

    2014-10-01

    Two triptycene-based ligands with pendant bromophenyl units have been prepared. These triptycene derivatives have been used as synthons for the synthesis of di and tri nuclear palladium complexes. The organic molecules and their corresponding organometallic complexes have been fully characterized using nuclear magnetic resonance (NMR), infrared (IR) spectroscopy and mass spectrometry. The mode of binding and effect of the complexes on pUC19 plasmid, calf thymus DNA and oligomer duplex DNA have been investigated by a host of analytical methods. The complexes brought about unwinding of supercoiled plasmid and the unwinding angle was found to be related to the binding affinity of the complexes with DNA, where both these parameters were guided by the structure of the complexes. Concentration-dependent inhibition of endonuclease activity of SspI and BamHI by the complexes indicates preference for G/C sequence for binding to DNA. However, neither the complexes did not introduce any cleavage at abasic site in oligomer duplex DNA, nor they created linear form of the plasmid upon co-incubation with the DNA samples. The interactions of the complexes with DNA were found to be strongly guided by the structure of the complexes, where intercalation as well as groove binding was observed, without inflicting any damage to the DNA. The mode of interaction of the complexes with DNA was further confirmed by isothermal calorimetry.

  2. The role of poly(ADP-ribose) in the DNA damage signaling network.

    Science.gov (United States)

    Malanga, Maria; Althaus, Felix R

    2005-06-01

    DNA damage signaling is crucial for the maintenance of genome integrity. In higher eukaryotes a NAD+-dependent signal transduction mechanism has evolved to protect cells against the genome destabilizing effects of DNA strand breaks. The mechanism involves 2 nuclear enzymes that sense DNA strand breaks, poly(ADP-ribose) polymerase-1 and -2 (PARP-1 and PARP-2). When activated by DNA breaks, these PARPs use NAD+ to catalyze their automodification with negatively charged, long and branched ADP-ribose polymers. Through recruitment of specific proteins at the site of damage and regulation of their activities, these polymers may either directly participate in the repair process or coordinate repair through chromatin unfolding, cell cycle progression, and cell survival-cell death pathways. A number of proteins, including histones, DNA topoisomerases, DNA methyltransferase-1 as well as DNA damage repair and checkpoint proteins (p23, p21, DNA-PK, NF-kB, XRCC1, and others) can be targeted in this manner; the interaction involves a specific poly(ADP-ribose)-binding sequence motif of 20-26 amino acids in the target domains.

  3. The Application of DNA-Biosensors and Differential Scanning Calorimetry to the Study of the DNA-Binding Agent Berenil

    Directory of Open Access Journals (Sweden)

    Marília O. F. Goulart

    2008-03-01

    Full Text Available The in situ DNA-damaging capacity of berenil (1 has been investigated usingan electrochemical approach employing double stranded (ds DNA-modified glassy carbonelectrode biosensors. Electrochemical voltammetric sensing of damage caused by 1 todsDNA was monitored by the appearance of peaks diagnostic of the oxidation of guanineand adenine. When 1 was incorporated directly onto the biosensor surface, DNA damagecould be observed at concentrations of additive as low as 10 μM. In contrast, when thedsDNA-modified biosensor was exposed to 1, in acetate buffer solution, the method wasmuch less sensitive and DNA damage could be detected only in the presence of 100 μMberenil. When mixed solutions of 1 and single stranded (ss DNA, polyguanylic acid orpolyadenylic acid were submitted to voltammetric study, the oxidation signals of therespective bases decreased in a concentration-dependent manner and the major variation ofthe adenine current peak indicated preferential binding of 1 to adenine. The electrochemical results were in close agreement with those deriving from a differentialscanning calorimetric study of the DNA-berenil complex.

  4. The Application of DNA-Biosensors and Differential Scanning Calorimetry to the Study of the DNA-Binding Agent Berenil.

    Science.gov (United States)

    De Abreu, Fabiane C; De Paula, Francine S; Ferreira, Danielle C M; Nascimento, Valberes B; Lopes, Julio C D; Santos, Alexandre M C; Santoro, Marcelo M; Salas, Carlos E; Goulart, Marília O F

    2008-03-03

    The in situ DNA-damaging capacity of berenil (1) has been investigated usingan electrochemical approach employing double stranded (ds) DNA-modified glassy carbonelectrode biosensors. Electrochemical voltammetric sensing of damage caused by 1 todsDNA was monitored by the appearance of peaks diagnostic of the oxidation of guanineand adenine. When 1 was incorporated directly onto the biosensor surface, DNA damagecould be observed at concentrations of additive as low as 10 μM. In contrast, when thedsDNA-modified biosensor was exposed to 1, in acetate buffer solution, the method wasmuch less sensitive and DNA damage could be detected only in the presence of 100 μMberenil. When mixed solutions of 1 and single stranded (ss) DNA, polyguanylic acid orpolyadenylic acid were submitted to voltammetric study, the oxidation signals of therespective bases decreased in a concentration-dependent manner and the major variation ofthe adenine current peak indicated preferential binding of 1 to adenine. The electrochemical results were in close agreement with those deriving from a differentialscanning calorimetric study of the DNA-berenil complex.

  5. Bacterial natural transformation by highly fragmented and damaged DNA

    DEFF Research Database (Denmark)

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

    2013-01-01

    of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations......DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often DNA is recognized as nutrient source...... for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake...

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

    Science.gov (United States)

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

    2016-04-01

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

  7. mapDamage: testing for damage patterns in ancient DNA sequences.

    Science.gov (United States)

    Ginolhac, Aurelien; Rasmussen, Morten; Gilbert, M Thomas P; Willerslev, Eske; Orlando, Ludovic

    2011-08-01

    Ancient DNA extracts consist of a mixture of contaminant DNA molecules, most often originating from environmental microbes, and endogenous fragments exhibiting substantial levels of DNA damage. The latter introduce specific nucleotide misincorporations and DNA fragmentation signatures in sequencing reads that could be advantageously used to argue for sequence validity. mapDamage is a Perl script that computes nucleotide misincorporation and fragmentation patterns using next-generation sequencing reads mapped against a reference genome. The Perl script outputs are further automatically processed in embedded R script in order to detect typical patterns of genuine ancient DNA sequences. The Perl script mapDamage is freely available with documentation and example files at http://geogenetics.ku.dk/all_literature/mapdamage/. The script requires prior installation of the SAMtools suite and R environment and has been validated on both GNU/Linux and MacOSX operating systems.

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

    Science.gov (United States)

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

    2014-12-11

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

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

    Science.gov (United States)

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

    2017-09-01

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

  10. DNA damage to spermatozoa has impacts on fertilization and pregnancy.

    Science.gov (United States)

    Lewis, S E M; Aitken, R J

    2005-10-01

    DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. The causes of this DNA damage are still uncertain but the major candidates are oxidative stress and aberrant apoptosis. The weight of evidence currently favours the former and, in keeping with this conclusion, positive results have been reported for antioxidant therapy both in vivo and in vitro. Resolving the causes of DNA damage in the male germ line will be essential if we are to prevent the generation of genetically damaged human embryos, particularly in the context of assisted conception therapy.

  11. Studies of the silencing of Baculovirus DNA binding protein

    OpenAIRE

    Quadt, I.; Lent, van, J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P1...

  12. DNA damage after intracerebroventricular injection of ouabain in rats.

    Science.gov (United States)

    Jornada, Luciano K; Valvassori, Samira S; Arent, Camila O; Leffa, Daniela; Damiani, Adriani A; Hainzenreder, Giana; Ferreira, Camila L; Moretti, Morgana; Andrade, Vanessa M; Quevedo, João

    2010-02-26

    There is an emerging body of data suggesting that bipolar disorder is associated with DNA damage. Intracerebroventricular (i.c.v.) administration of ouabain in rats results in manic-like alterations. We evaluated DNA damage of peripheral blood, cerebrospinal fluid and hippocampus of rats after i.c.v. ouabain injection. Ouabain-induced hyperlocomotion was examined in an open field. Additionally, we used single cell gel electrophoresis (comet assay) to measure early transient damage in cerebrospinal fluid (CSF), hippocampus and blood; and the micronucleus test to measure persistent damage in total blood samples of rats after ouabain administration. Our findings demonstrated that ouabain induced hyperlocomotion in rats, and this response remained up to 7 days following a single i.c.v. injection. In addition, we observed that the persistent increase in the rat spontaneous locomotion is associated with increased hippocampal and peripheral index of early DNA damage in rats. No significant alterations were observed in the micronucleus frequency in total blood samples of the rats after the ouabain i.c.v. injection. These results suggest that ouabain may induce peripheral and central early DNA damage, but this early damage may be repaired.

  13. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that f

  14. DNA Damage Caused By Pesticide-contaminated Soil

    Institute of Scientific and Technical Information of China (English)

    K.KRISHNAMURTHI; S. SARAVANA DEVI; T. CHAKRABARTI

    2006-01-01

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

  15. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH2 groups (CONH2-SWNT) exhibited very strong interactions between the CONH2-SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  16. Posttranscriptional regulation of gene expression—adding another layer of complexity to the DNA damage response

    Science.gov (United States)

    Boucas, Jorge; Riabinska, Arina; Jokic, Mladen; Herter-Sprie, Grit S.; Chen, Shuhua; Höpker, Katja; Reinhardt, H. Christian

    2012-01-01

    In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components—a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases. PMID:22936947

  17. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    Directory of Open Access Journals (Sweden)

    Marie-Jo Halaby

    2015-01-01

    Full Text Available Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES that is located at the 5′-untranslated region (UTR of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80 has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress.

  18. Detection of DNA damage induced by topoisomerase II inhibitors, gamma radiation and crosslinking agents using the comet assay.

    Science.gov (United States)

    Hazlehurst, Lori A

    2009-01-01

    The comet assay is a simple gel electrophoresis method for visualizing and quantifying DNA damage. The comet assay is sensitive and reproducible and can be used to detect single-strand DNA breaks, double-strand DNA breaks, protein-associated DNA strand breaks and DNA crosslinks. The comet assay uses fluorescent DNA-binding dyes to detect both damaged DNA that resides in the tail region and undamaged DNA that is retained in the head region following gel electrophoresis. This assay is a single cell-based assay and thus is highly adaptable for measuring DNA damage in clinical samples. Furthermore, unlike other assays the detection of DNA damage is not dependent on the random incorporation of radiolabeled nucleotides. Again this can be problematic with clinical samples as proliferation rates are often slow and culturing of primary patient specimens for 48 h required to randomly label DNA is often not possible. In this chapter we will outline the comet assay for the detection of DNA damage induced by topoisomerase II inhibitors, cross-linking agents and gamma radiation.

  19. DNA damage, repair and tanning acceleration

    NARCIS (Netherlands)

    Vink, A.A.; Berg, P.T.M. van den; Roza, L.

    1999-01-01

    Exposure of the skin to solar ultraviolet radiation (UV) leads to various adverse effects, such as the induction of cellular damage and mutations, suppression of the skin's immune system, and the induction of skin cancer. These effects are the consequence of various molecular alterations in the skin

  20. APOBEC3A damages the cellular genome during DNA replication.

    Science.gov (United States)

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

    2016-01-01

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

  1. Catalytic and non-catalytic roles for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response.

    Directory of Open Access Journals (Sweden)

    Christina L Stallings

    Full Text Available Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways (HR, NHEJ, SSA, and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance.

  2. Catalytic and non-catalytic roles for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response.

    Science.gov (United States)

    Stallings, Christina L; Chu, Linda; Li, Lucy X; Glickman, Michael S

    2011-01-01

    Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs) differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways (HR, NHEJ, SSA), and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance.

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

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

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

  4. A novel role of the chromokinesin Kif4A in DNA damage response.

    Science.gov (United States)

    Wu, Guikai; Zhou, Longen; Khidr, Lily; Guo, Xuning Emily; Kim, Wankee; Lee, Young Mi; Krasieva, Tatiana; Chen, Phang-Lang

    2008-07-01

    Chromokinesins are microtubule-motor molecules that possess chromatin binding activity and are important for mitotic and meiotic regulation. The chromokinesin-member Kif4A is unique in that it localizes to nucleus during interphase of the cell cycle. Kif4 deletion by gene targeting in mouse embryonic cells was known to associate with DNA damage response. However, its precise role in DNA damage or repair pathway is not clear. Here we report that Kif4A associates with BRCA2 in a biochemical identification and that the interaction is mediated by the Kif4A C-terminal cargo-binding domain and BRCA2 C-terminal conserved region. Upon nucleus-specific laser micro-irradiation, Kif4A was rapidly recruited to sites of DNA damage. Significantly, the depletion of Kif4A from cells by shRNA impaired the ionizing-radiation induced foci (IRIF) formation of Rad51, both quantitatively and qualitatively. In contrast, the IRIF of gamma-H2AX or NBS1 was largely intact. Moreover, Kif4A knockdown rendered cells hypersensitive to ionizing radiation in a colonogenic survival assay. We further demonstrated that Kif4A deficiency led to significantly decreased homologous recombination in an I-SceI endonuclease induced in vivo recombination assay. Together, our results suggest a novel role for a chromokinesin family member in the DNA damage response by modulating the BRCA2/Rad51 pathway.

  5. RNA recognition by the DNA end-binding Ku heterodimer.

    Science.gov (United States)

    Dalby, Andrew B; Goodrich, Karen J; Pfingsten, Jennifer S; Cech, Thomas R

    2013-06-01

    Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

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

    Directory of Open Access Journals (Sweden)

    Nicole Schupp

    2016-01-01

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

  7. Damaging the Integrated HIV Proviral DNA with TALENs.

    Directory of Open Access Journals (Sweden)

    Christy L Strong

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

  8. Characteristics of DNA replication in isolated nuclei initiated by an aprotinin-binding protein.

    Science.gov (United States)

    Coffman, F D; Fresa, K L; Hameed, M; Cohen, S

    1993-02-01

    Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase alpha (a principal polymerase in DNA replication) but that DNA polymerase beta (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of 3H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events.

  9. Extended HSR/CARD domain mediates AIRE binding to DNA

    Energy Technology Data Exchange (ETDEWEB)

    Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-12-25

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

  10. Oxidative stress and DNA damages induced by cadmium accumulation

    Institute of Scientific and Technical Information of China (English)

    LIN Ai-jun; ZHANG Xu-hong; CHEN Mei-mei; CAO Qing

    2007-01-01

    Experimental evidence shows that cadmium (Cd) could induce oxidative stress and then causes DNA damage in animal cells, however, whether such effect exists in plants is still unclear. In the present study, Vicia faba plants was exposed to 5 and 10 mg/L Cd for 4 d to investigate the distribution of Cd in plant, the metal effects on the cell lipids, antioxidative enzymes and DNA damages in leaves. Cd induced an increase in Cd concentrations in plants. An enhanced level of lipid peroxidation in leaves and an enhanced concentration of H2O2 in root tissues suggested that Cd caused oxidative stress in Vicia faba. Compared with control, Cd-induced enhancement in superoxide dismutase activity was significant at 5 mg/L than at 10 mg/kg in leaves, by contrast, catalase and peroxidaseactivities were significantly suppressed by Cd addition. DNA damage was detected by neutral/neutral, alkaline/neutral and alkaline/alkaline Comet assay. Increased levels of DNA damages induced by Cd occurred with reference to oxidative stress in leaves, therefore, oxidative stress induced by Cd accumulation in plants contributed to DNA damages and was possibly an important mechanism of Cd-phytotoxicity in Vicia faba plants.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    Science.gov (United States)

    Laspia, M F; Wallace, S S

    1989-05-05

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

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

    NARCIS (Netherlands)

    S. Vives (Sergi); M.T. Gilbert (Thomas); C. Arenas (Conchita); E. Gigli (Elena); O. Lao Grueso (Oscar); C. Lalueza-Fox (Carles)

    2008-01-01

    textabstractBackground. We have analysed the distribution of post mortem DNA damage derived miscoding lesions from the datasets of seven published Neandertal specimens that have extensive cloned sequence coverage over the mitochondrial DNA (mtDNA) hypervariable region 1 (HVS1). The analysis was rest

  14. Genomic instability and DNA damage responses in progeria arising from defective maturation of prelamin A.

    Science.gov (United States)

    Musich, Phillip R; Zou, Yue

    2009-01-01

    Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for aLMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component.

  15. Parvovirus diversity and DNA damage responses.

    Science.gov (United States)

    Cotmore, Susan F; Tattersall, Peter

    2013-02-01

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

  16. Evaluation of DNA damage using microwave dielectric absorption spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-12-15

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

  17. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    Science.gov (United States)

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

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

    Science.gov (United States)

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

    2016-07-26

    We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1(f/f)p53(f/f)), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors.

  19. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-11-22

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

  20. A dual role of Cdk2 in DNA damage response

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2009-05-01

    Full Text Available Abstract Once it was believed that Cdk2 was the master regulator of S phase entry. Gene knockout mouse studies of cell cycle regulators revealed that Cdk2 is dispensable for S phase initiation and progression whereby Cdk1 can compensate for the loss of Cdk2. Nevertheless, recent evidence indicates that Cdk2 is involved in cell cycle independent functions such as DNA damage repair. Whether these properties are unique to Cdk2 or also being compensated by other Cdks in the absence of Cdk2 is under extensive investigation. Here we review the emerging new role of Cdk2 in DNA damage repair and also discuss how the loss of Cdk2 impacts the G1/S phase DNA damage checkpoint.

  1. A dual role of Cdk2 in DNA damage response.

    Science.gov (United States)

    Satyanarayana, Ande; Kaldis, Philipp

    2009-05-18

    Once it was believed that Cdk2 was the master regulator of S phase entry. Gene knockout mouse studies of cell cycle regulators revealed that Cdk2 is dispensable for S phase initiation and progression whereby Cdk1 can compensate for the loss of Cdk2. Nevertheless, recent evidence indicates that Cdk2 is involved in cell cycle independent functions such as DNA damage repair. Whether these properties are unique to Cdk2 or also being compensated by other Cdks in the absence of Cdk2 is under extensive investigation. Here we review the emerging new role of Cdk2 in DNA damage repair and also discuss how the loss of Cdk2 impacts the G1/S phase DNA damage checkpoint.

  2. Increased DNA damage and oxidative stress among silver jewelry workers.

    Science.gov (United States)

    Aktepe, Necmettin; Kocyigit, Abdurrahim; Yukselten, Yunus; Taskin, Abdullah; Keskin, Cumali; Celik, Hakim

    2015-04-01

    Silver has long been valued as a precious metal, and it is used to make ornaments, jewelry, high-value tableware, utensils, and currency coins. Human exposures to silver and silver compounds can occur oral, dermal, or by inhalation. In this study, we investigated genotoxic and oxidative effects of silver exposure among silver jewelry workers. DNA damage in peripheral mononuclear leukocytes was measured by using the comet assay. Serum total antioxidative status (TAS), total oxidative status (TOS), total thiol contents, and ceruloplasmin levels were measured by using colorimetric methods among silver jewelry workers. Moreover, oxidative stress index (OSI) was calculated. Results were compared with non-exposed healthy subjects. The mean values of mononuclear leukocyte DNA damage were significantly higher than control subjects (p jewelry workers caused oxidative stress and accumulation of severe DNA damage.

  3. Aptamer-Binding Directed DNA Origami Pattern for Logic Gates.

    Science.gov (United States)

    Yang, Jing; Jiang, Shuoxing; Liu, Xiangrong; Pan, Linqiang; Zhang, Cheng

    2016-12-14

    In this study, an aptamer-substrate strategy is introduced to control programmable DNA origami pattern. Combined with DNA aptamer-substrate binding and DNAzyme-cutting, small DNA tiles were specifically controlled to fill into the predesigned DNA origami frame. Here, a set of DNA logic gates (OR, YES, and AND) are performed in response to the stimuli of adenosine triphosphate (ATP) and cocaine. The experimental results are confirmed by AFM imaging and time-dependent fluorescence changes, demonstrating that the geometric patterns are regulated in a controllable and programmable manner. Our approach provides a new platform for engineering programmable origami nanopatterns and constructing complex DNA nanodevices.

  4. Hydrogen sulfide induces oxidative damage to RNA and DNA in a sulfide-tolerant marine invertebrate.

    Science.gov (United States)

    Joyner-Matos, Joanna; Predmore, Benjamin L; Stein, Jenny R; Leeuwenburgh, Christiaan; Julian, David

    2010-01-01

    Hydrogen sulfide acts as an environmental toxin across a range of concentrations and as a cellular signaling molecule at very low concentrations. Despite its toxicity, many animals, including the mudflat polychaete Glycera dibranchiata, are periodically or continuously exposed to sulfide in their environment. We tested the hypothesis that a broad range of ecologically relevant sulfide concentrations induces oxidative stress and oxidative damage to RNA and DNA in G. dibranchiata. Coelomocytes exposed in vitro to sulfide (0-3 mmol L(-1) for 1 h) showed dose-dependent increases in oxidative stress (as 2',7'-dichlorofluorescein fluorescence) and superoxide production (as dihydroethidine fluorescence). Coelomocytes exposed in vitro to sulfide (up to 0.73 mmol L(-1) for 2 h) also acquired increased oxidative damage to RNA (detected as 8-oxo-7,8-dihydroguanosine) and DNA (detected as 8-oxo-7,8-dihydro-2'-deoxyguanosine). Worms exposed in vivo to sulfide (0-10 mmol L(-1) for 24 h) acquired elevated oxidative damage to RNA and DNA in both coelomocytes and body wall tissue. While the consequences of RNA and DNA oxidative damage are poorly understood, oxidatively damaged deoxyguanosine bases preferentially bind thymine, causing G-T transversions and potentially causing heritable point mutations. This suggests that sulfide can be an environmental mutagen in sulfide-tolerant invertebrates.

  5. Increased Mutagen Sensitivity and DNA Damage in Pulmonary Arterial Hypertension

    Science.gov (United States)

    Federici, Chiara; Drake, Kylie M.; Rigelsky, Christina M.; McNelly, Lauren N.; Meade, Sirena L.; Comhair, Suzy A. A.; Erzurum, Serpil C.

    2015-01-01

    Rationale: Pulmonary arterial hypertension (PAH) is a serious lung condition characterized by vascular remodeling in the precapillary pulmonary arterioles. We and others have demonstrated chromosomal abnormalities and increased DNA damage in PAH lung vascular cells, but their timing and role in disease pathogenesis is unknown. Objectives: We hypothesized that if DNA damage predates PAH, it might be an intrinsic cell property that is present outside the diseased lung. Methods: We measured DNA damage, mutagen sensitivity, and reactive oxygen species (ROS) in lung and blood cells from patients with Group 1 PAH, their relatives, and unrelated control subjects. Measurements and Main Results: Baseline DNA damage was significantly elevated in PAH, both in pulmonary artery endothelial cells (P < 0.05) and peripheral blood mononuclear cells (PBMC) (P < 0.001). Remarkably, PBMC from unaffected relatives showed similar increases, indicating this is not related to PAH treatments. ROS levels were also higher (P < 0.01). DNA damage correlated with ROS production and was suppressed by antioxidants (P < 0.001). PBMC from patients and relatives also showed markedly increased sensitivity to two chemotherapeutic drugs, bleomycin and etoposide (P < 0.001). Results were consistent across idiopathic, heritable, and associated PAH groups. Conclusions: Levels of baseline and mutagen-induced DNA damage are intrinsically higher in PAH cells. Similar results in PBMC from unaffected relatives suggest this may be a genetically determined trait that predates disease onset and may act as a risk factor contributing to lung vascular remodeling following endothelial cell injury. Further studies are required to fully characterize mutagen sensitivity, which could have important implications for clinical management. PMID:25918951

  6. DNA damage and oxidative status in PFAPA syndrome.

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

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

    2015-05-01

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

  8. Mechanisms and Components of the DNA Damage Checkpoint

    Science.gov (United States)

    2002-09-01

    Saccharomyces cerevisiae DNA damage checkpoint. Molecular Cell 9: 1055-1065. (reprint included as Appendix 2) "* Schwartz, M.F., Duong, J.K., Sun, Z., Pradhan...phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. Molecular Cell 9, 1055-1065. 13 Molecular Cell , Vol. 9,1055-1065...Cambridge, Massachusetts 02139. 1999), and mutation of conserved amino acids in the Molecular Cell 1056 A Rad9 B ,•o 0, 1 sitesN NC -T6 RVTQSA o- 0~ --T240

  9. Nuclear DNA damage signalling to mitochondria in ageing.

    Science.gov (United States)

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

    2016-05-01

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

  10. Linking abnormal mitosis to the acquisition of DNA damage

    Science.gov (United States)

    Pellman, David

    2012-01-01

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

  11. 8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage

    DEFF Research Database (Denmark)

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

    1993-01-01

    , rats, and mice. The excretion of 8OHdG decreased with age in rats in parallel with the decline in metabolic rate with advancing age. The excretion of 8OHdG reflects the formation and repair of only one out of approximately 20 described oxidative DNA modifications. So far, methods are not available......Living organisms are continuously exposed to reactive oxygen species as a consequence of biochemical reactions as well as external factors. Oxidative DNA damage has been implicated in aging, carcinogenesis and other degenerative diseases. The urinary excretion of the DNA repair product 8......-hydroxydeoxyguanosine (8OHdG) has been proposed as a noninvasive biomarker of oxidative DNA damage in humans in vivo. We have developed a three-dimensional HPLC analysis with electrochemical detection for the analysis of 8OHdG in urine and studied factors affecting the excretion of this biomarker in 83 healthy humans...

  12. DNA damage response in nephrotoxic and ischemic kidney injury.

    Science.gov (United States)

    Yan, Mingjuan; Tang, Chengyuan; Ma, Zhengwei; Huang, Shuang; Dong, Zheng

    2016-10-27

    DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore relies on a thorough elucidation of the DDR pathways in various forms of AKI.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-08-01

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

  14. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    Science.gov (United States)

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  15. Visually Relating Gene Expression and in vivo DNA Binding Data

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

    2011-09-20

    Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

  16. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways.

    Science.gov (United States)

    Ichijima, Yosuke; Sin, Ho-Su; Namekawa, Satoshi H

    2012-08-01

    Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.

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

    Science.gov (United States)

    Mullenders, Leon

    2015-12-01

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

  18. Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

    Energy Technology Data Exchange (ETDEWEB)

    Clement, Flurina C.; Camenisch, Ulrike; Fei, Jia; Kaczmarek, Nina; Mathieu, Nadine [Institute of Pharmacology and Toxicology, University of Zuerich-Vetsuisse, Winterthurerstrasse 260, CH-8057 Zuerich (Switzerland); Naegeli, Hanspeter, E-mail: naegelih@vetpharm.uzh.ch [Institute of Pharmacology and Toxicology, University of Zuerich-Vetsuisse, Winterthurerstrasse 260, CH-8057 Zuerich (Switzerland)

    2010-03-01

    The recognition and subsequent repair of DNA damage are essential reactions for the maintenance of genome stability. A key general sensor of DNA lesions is xeroderma pigmentosum group C (XPC) protein, which recognizes a wide variety of helix-distorting DNA adducts arising from ultraviolet (UV) radiation, genotoxic chemicals and reactive metabolic byproducts. By detecting damaged DNA sites, this unique molecular sensor initiates the global genome repair (GGR) pathway, which allows for the removal of all the aforementioned lesions by a limited repertoire of excision factors. A faulty GGR activity causes the accumulation of DNA adducts leading to mutagenesis, carcinogenesis, neurological degeneration and other traits of premature aging. Recent findings indicate that XPC protein achieves its extraordinary substrate versatility by an entirely indirect readout strategy implemented in two clearly discernible stages. First, the XPC subunit uses a dynamic sensor interface to monitor the double helix for the presence of non-hydrogen-bonded bases. This initial screening generates a transient nucleoprotein intermediate that subsequently matures into the ultimate recognition complex by trapping undamaged nucleotides in the abnormally oscillating native strand, in a way that no direct contacts are made between XPC protein and the offending lesion itself. It remains to be elucidated how accessory factors like Rad23B, centrin-2 or the UV-damaged DNA-binding complex contribute to this dynamic two-stage quality control process.

  19. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    Energy Technology Data Exchange (ETDEWEB)

    Crissman, Harry A.; Cui, H. H. (H. Helen); Steinkamp, J. A.

    2002-01-01

    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

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

    Directory of Open Access Journals (Sweden)

    Hamidreza Namazi

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yan Chen

    2014-10-01

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

  2. Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes.

    Directory of Open Access Journals (Sweden)

    Masashi Fujita

    Full Text Available Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4 photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4 photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

  3. Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

    Energy Technology Data Exchange (ETDEWEB)

    De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K. (VCU); (Mount Sinai Hospital)

    2013-11-20

    Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.

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

    Directory of Open Access Journals (Sweden)

    Katarzyna Zaremba-Niedźwiedzka

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

  5. Dissociation Dynamics of XPC-RAD23B from Damaged DNA Is a Determining Factor of NER Efficiency.

    Directory of Open Access Journals (Sweden)

    Benjamin Hilton

    Full Text Available XPC-RAD23B (XPC plays a critical role in human nucleotide excision repair (hNER as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency. We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER.

  6. DNA damage under simulated extraterrestrial conditions in bacteriophage T7

    Science.gov (United States)

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

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

  7. DNA-binding specificities of human transcription factors.

    Science.gov (United States)

    Jolma, Arttu; Yan, Jian; Whitington, Thomas; Toivonen, Jarkko; Nitta, Kazuhiro R; Rastas, Pasi; Morgunova, Ekaterina; Enge, Martin; Taipale, Mikko; Wei, Gonghong; Palin, Kimmo; Vaquerizas, Juan M; Vincentelli, Renaud; Luscombe, Nicholas M; Hughes, Timothy R; Lemaire, Patrick; Ukkonen, Esko; Kivioja, Teemu; Taipale, Jussi

    2013-01-17

    Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lavin, Martin F. [Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Brisbane 4029 (Australia) and Central Clinical Division, University of Queensland, Brisbane (Australia)]. E-mail: martinl@qimr.edu.au; Birrell, Geoff [Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Brisbane 4029 (Australia); Chen, Philip [Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Brisbane 4029 (Australia); Kozlov, Sergei [Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Brisbane 4029 (Australia); Scott, Shaun [Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Brisbane 4029 (Australia); Gueven, Nuri [Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Brisbane 4029 (Australia)

    2005-01-06

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

  9. Roles of histone ubiquitylation in DNA damage signaling

    Institute of Scientific and Technical Information of China (English)

    Sui-Sui DONG; Michael S. Y. HUEN

    2011-01-01

    Histone ubiquitylation has emerged as an important chromatin modification associated with DNA damage signaling and repair pathways.These histone marks,laid down by E3 ubiquitin ligases that include RNF8 and RNF168,decorate chromatin domains surrounding DNA double-strand breaks (DSBs).Recent work implicated ubiquitylated histones in orchestrating cell cycle checkpoints,DNA repair and gene transcription.Here we summarize recent advances that contribute to our current knowledge of the highly dynamic nature of DSB-associated histone ubiquitylation,and discuss major challenges ahead in understanding the versatility of ubiquitin conjugation in maintaining genome stability.

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

    Science.gov (United States)

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

    1989-02-01

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

  11. DNA damage by carbonyl stress in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-01-28

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

  12. A mitotic phosphorylation feedback network connects Cdk1, Plk1, 53BP1, and Chk2 to inactivate the G(2)/M DNA damage checkpoint

    DEFF Research Database (Denmark)

    van Vugt, Marcel A T M; Gardino, Alexandra K; Linding, Rune;

    2010-01-01

    the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation......DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular...... of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity...

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

    DEFF Research Database (Denmark)

    Møller, P; Loft, S; Lundby, C

    2001-01-01

    ; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage....... Urinary excretion of 8-oxodG increased during the first day in altitude hypoxia, and there were more endonuclease III-sensitive sites on day 3 at high altitude. The subjects had more DNA strand breaks in altitude hypoxia than at sea level. The level of DNA strand breaks further increased immediately after...... exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive...

  14. CtIP-dependent DNA resection is required for DNA damage checkpoint maintenance but not initiation

    DEFF Research Database (Denmark)

    Kousholt, Arne Nedergaard; Fugger, Kasper; Hoffmann, Saskia

    2012-01-01

    by camptothecin and ionizing radiation. In contrast, we find that DNA end resection was critically required for sustained ATR-CHK1 checkpoint signaling and for maintaining both the intra-S- and G2-phase checkpoints. Consequently, resection-deficient cells entered mitosis with persistent DNA damage. In conclusion......To prevent accumulation of mutations, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homology-directed repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of ATR (ataxia telangiectasia...... mutated and Rad3 related) and CHK1 kinases to induce the cell cycle checkpoint. In this paper, we show that CHK1 was rapidly and robustly activated before detectable end resection. Moreover, we show that the key resection factor CtIP was dispensable for initial ATR-CHK1 activation after DNA damage...

  15. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

  16. Human single-stranded DNA binding proteins: guardians of genome stability

    Institute of Scientific and Technical Information of China (English)

    Yuanzhong Wu; Jinping Lu; Tiebang Kang

    2016-01-01

    Single-stranded DNA-binding proteins (SSBs) are essential for maintaining the integrity of the genome in all organisms.All processes related to DNA,such as replication,excision,repair,and recombination,require the participation of SSBs whose oligonucleotideaoligosaccharide-binding (OB)-fold domain is responsible for the interaction with single-stranded DNA (ssDNA).For a long time,the heterotrimeric replication protein A (RPA) complex was believed to be the only nuclear SSB in eukanyotes to participate in ssDNA processing,while mitochondrial SSBs that are consewed with prokaryotic SSBs were shown to be essential for maintaining genome stability in eukaryotic mitochondria.In recent years,two new proteins,hSSB1 and hSSB2 (human SSBs 1/2),were identified and have better sequence similarity to bacterial and archaeal SSBs than RPA.This review summarizes the current understanding of these human SSBs in DNA damage repair and in cell-cycle checkpoint activation following DNA damage,as well as their relationships with cancer.

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

    Science.gov (United States)

    Luftig, Micah A

    2014-11-01

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

  18. Thermodynamics of cationic lipid binding to DNA and DNA condensation: roles of electrostatics and hydrophobicity.

    Science.gov (United States)

    Matulis, Daumantas; Rouzina, Ioulia; Bloomfield, Victor A

    2002-06-26

    Alkylammonium binding to DNA was studied by isothermal titration calorimetry. Experimental data, obtained as functions of alkyl chain length, salt concentration, DNA concentration, and temperature, provided a detailed thermodynamic description of lipid-DNA binding reactions leading to DNA condensation. Lipid binding, counterion displacement, and DNA condensation were highly cooperative processes, driven by a large increase in entropy and opposed by a relatively small endothermic enthalpy at room temperature. Large negative heat capacity change indicated a contribution from hydrophobic interactions between aliphatic tails. An approximation of lipid-DNA binding as dominated by two factors-ionic and hydrophobic interactions-yielded a model that was consistent with experimental data. Chemical group contributions to the energetics of binding were determined and could be used to predict energetics of other lipid binding to DNA. Electrostatic and hydrophobic contributions to Gibbs free energy, enthalpy, entropy, and heat capacity could be distinguished by applying additivity principles. Binding of lipids with two, three, and four aliphatic tails was investigated and compared to single-tailed lipid binding. Structurally, the model suggests that lipid cationic headgroups and aliphatic tails distribute evenly and lay down on DNA surface without the formation of micelles.

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

    DEFF Research Database (Denmark)

    Jackson, Stephen P; Bartek, Jiri

    2009-01-01

    , signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management....

  20. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    Science.gov (United States)

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

  1. UV Radiation Damage and Bacterial DNA Repair Systems

    Science.gov (United States)

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

    2006-01-01

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

  2. Mechanism study of goldenseal-associated DNA damage.

    Science.gov (United States)

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

    2013-07-31

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

  3. 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.

  4. 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.

  5. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication.

    Science.gov (United States)

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the

  6. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  7. Antineoplastic DNA-binding compounds: intercalating and minor groove binding drugs.

    Science.gov (United States)

    Mišković, Katarina; Bujak, Maro; Baus Lončar, Mirela; Glavaš-Obrovac, Ljubica

    2013-12-01

    DNA intercalating and minor groove binding compounds are new weapons in the battle against malignant diseases. These antineoplastic agents target the DNA molecule and interfere with the cell cycle leading to rapidly proliferating cell death. They are mainly derivates of a naturally occurring organic compound derived from a microorganism or plant. Intercalators usually act as topoisomerase I and/or II poisons, while the mechanisms of DNA minor groove binders are a combination of several steps including topoisomerase poisoning. This paper gives an overview of some of the developed DNA intercalating and minor groove binding compounds, as well as an explanation of their chemical structures, origins, and application in chemotherapy.

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

    DEFF Research Database (Denmark)

    Jorgensen, Anders; Krogh, Jesper; Miskowiak, Kamilla;

    2013-01-01

    , such as dementia and type 2 diabetes. We hypothesized that increased severity of depression is associated with increased systemic oxidatively generated DNA and RNA damage, and that this increase is attenuated by an effective antidepressant treatment. METHODS: The urinary excretion of markers of systemic......BACKGROUND: Depression has been associated with increased oxidative stress and hypothesized to accelerate aging. Nucleic acid damage from oxidation is a critical part of the aging process, and a suggested early event in age-related somatic morbidities that are also prevalent in depression...... oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy...

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

    CERN Document Server

    Hill, M A

    1999-01-01

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

  10. Oxidative DNA Damage in Neurons: Implication of Ku in Neuronal Homeostasis and Survival

    Directory of Open Access Journals (Sweden)

    Daniela De Zio

    2012-01-01

    Full Text Available Oxidative DNA damage is produced by reactive oxygen species (ROS which are generated by exogenous and endogenous sources and continuously challenge the cell. One of the most severe DNA lesions is the double-strand break (DSB, which is mainly repaired by nonhomologous end joining (NHEJ pathway in mammals. NHEJ directly joins the broken ends, without using the homologous template. Ku70/86 heterodimer, also known as Ku, is the first component of NHEJ as it directly binds DNA and recruits other NHEJ factors to promote the repair of the broken ends. Neurons are particularly metabolically active, displaying high rates of transcription and translation, which are associated with high metabolic and mitochondrial activity as well as oxygen consumption. In such a way, excessive oxygen radicals can be generated and constantly attack DNA, thereby producing several lesions. This condition, together with defective DNA repair systems, can lead to a high accumulation of DNA damage resulting in neurodegenerative processes and defects in neurodevelopment. In light of recent findings, in this paper, we will discuss the possible implication of Ku in neurodevelopment and in mediating the DNA repair dysfunction observed in certain neurodegenerations.

  11. Polycomb protein EZH2 regulates cancer cell fate decision in response to DNA damage.

    Science.gov (United States)

    Wu, Z; Lee, S T; Qiao, Y; Li, Z; Lee, P L; Lee, Y J; Jiang, X; Tan, J; Aau, M; Lim, C Z H; Yu, Q

    2011-11-01

    Polycomb protein histone methyltransferase enhancer of Zeste homologe 2 (EZH2) is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating DNA damage response. Here, we show that EZH2 is an important determinant of cell fate decision in response to genotoxic stress. EZH2 depletion results in abrogation of both cell cycle G1 and G2/M checkpoints, directing DNA damage response toward predominant apoptosis in both p53-proficient and p53-deficient cancer cells, but not in normal cells. Mechanistically, EZH2 regulates DNA damage response in p53 wild-type cells mainly through transcriptional repression of FBXO32, which binds to and directs p21 for proteasome-mediated degradation, whereas it affects p53-deficient cells through regulating Chk1 activation by a distinct mechanism. Furthermore, pharmacological depletion of EZH2 phenocopies the effects of EZH2 knockdown on cell cycle checkpoints and apoptosis. These data unravel a crucial role of EZH2 in determining the cancer cell outcome following DNA damage and suggest that therapeutic targeting oncogenic EZH2 might serve as a strategy for improving conventional chemotherapy in a given malignancy.

  12. Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome.

    Science.gov (United States)

    Iyama, Teruaki; Wilson, David M

    2016-01-16

    Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.

  13. Regulation of DNA replication by the S-phase DNA damage checkpoint

    Directory of Open Access Journals (Sweden)

    Rhind Nicholas

    2009-07-01

    Full Text Available Abstract Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM. Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    with the DNA damaging agent methyl methanesulfonate (MMS). We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified......Small ubiquitin-like modifiers play critical roles in the DNA damage response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach in order to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment...... dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO...

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

    DEFF Research Database (Denmark)

    Jørgensen, Anders

    2013-01-01

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

  16. DNA damage by reactive species: Mechanisms, mutation and repair.

    Science.gov (United States)

    Jena, N R

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA-protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA-protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.

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

    Science.gov (United States)

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

    2011-08-01

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

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

    Science.gov (United States)

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

    2008-01-01

    In the last decade, the Comet assay has been used increasingly in studies of workers potentially exposed to genotoxic substances in the workplace or environment. Significant increases in DNA damage measured with the Comet assay has been reported in lymphocytes of agricultural workers; however, less intrusive means of biomonitoring are needed in epidemiological investigations. This study was designed to use the Comet assay to describe the association of markers of DNA damage in oral leukocytes with biomarkers of pesticide exposure in 134 farmworkers working in berry crops in Oregon compared to control populations. The authors also examined the extent of DNA damage in young workers compared to adults and the effect of work histories, lifestyle factors, and diet on markers of DNA damage. Urinary levels of organophosphate pesticides were low at the time of sampling; however, mean levels of the Captan metabolite tetrahydrophthalimide (THPI) were found to be shifted significantly higher in the farmworkers (0.14 microg/ml) compared to controls (0.078 microg/ml) (one-sided p value=.01). Likewise, the combined molar equivalent of all dialkylphosphate metabolites was marginally higher in farmworkers (p value=.05). The mean tail intensity was significantly greater for agricultural workers compared to controls (one-sided p valuedamage in the oral leukocytes. On average, the mean tail intensity was 10.9 units greater for agricultural workers (95% CI: 6-16 units greater). Tail moment was also significantly greater for agricultural workers compared to nonagricultural workers (one-sided p valuepesticide types on DNA damage and to capture the temporal relationship between exposure to agricultural chemicals and changes in Comet parameters.

  19. Binding Isotherms and Cooperative Effects for Metal-DNA Complexes

    CERN Document Server

    Gelagutashvili, Eteri

    2008-01-01

    The stoichiometric binding constants of Nickel(II), Cobalt(II), Manganese(II), Silver(I), Zinc(II) ions with DNA, from Spirulina platensis were determined from their binding isotherms by equilibrium dialysis and atomic absorption spectroscopy. It was shown, that the nature of these ions interaction with DNA, from S .platensis is different. For Cobalt(II), Zinc(II) ions were observed cooperative effects and existence of two different types of the binding sites. Nickel(II)_, Silver(I) -DNA complexes shows independent and identical binding sites and Manganese(II)_ negative cooperative interaction. The logarithm of binding constants for Cobalt (II)_, Nickel (II)_, Manganese (II)_, Zinc (II)_, Silver (I) - DNA, from S. platensis in 3 mM Na(I) are 5.11; 5.18; 4.77; 5.05; 5.42; respectively. The linear correlation of logarithm of binding constants (for complexes of metal-DNA from S. platensis) and the covalent index of Pauling are observed.

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

    Directory of Open Access Journals (Sweden)

    Ana Belén Herrero

    2017-05-01

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

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

    Science.gov (United States)

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

    2016-05-18

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

  2. DNA Damage: A Main Determinant of Vascular Aging

    Directory of Open Access Journals (Sweden)

    Paula K. Bautista-Niño

    2016-05-01

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

  3. Effect of clustered peptide binding on DNA condensation.

    Science.gov (United States)

    Haley, Jennifer; Kabiru, Paul; Geng, Yan

    2010-01-01

    DNA condensation in-vitro has been studied as a model system to reveal common principles underlying gene packaging in biology, and as the critical first step towards the development of non-viral gene delivery vectors. In this study, we use a bio-inspired approach, where small DNA-binding peptides are controllably clustered by an amphiphilic block copolymer scaffold, to reveal the effect of clustered peptide binding on the energetics, size, shape and physical properties of DNA condensation in-vitro. This provides insights into the general architectural effect of gene-binding proteins on DNA condensation process. Moreover, the versatility afforded by regulating the clustering density and composition of peptides may provide a novel design platform for gene delivery applications in the future.

  4. 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

    damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase......-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors....

  5. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

    Directory of Open Access Journals (Sweden)

    Molly L. Bristol

    2016-06-01

    Full Text Available Human papillomaviruses (HPVs are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1, does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

  6. Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

    Science.gov (United States)

    Panat, Niranjan A; Singh, Beena G; Maurya, Dharmendra K; Sandur, Santosh K; Ghaskadbi, Saroj S

    2016-05-05

    Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

  7. Role of Nicotinamide in DNA Damage, Mutagenesis, and DNA Repair

    Directory of Open Access Journals (Sweden)

    Devita Surjana

    2010-01-01

    Full Text Available Nicotinamide is a water-soluble amide form of niacin (nicotinic acid or vitamin B3. Both niacin and nicotinamide are widely available in plant and animal foods, and niacin can also be endogenously synthesized in the liver from dietary tryptophan. Nicotinamide is also commercially available in vitamin supplements and in a range of cosmetic, hair, and skin preparations. Nicotinamide is the primary precursor of nicotinamide adenine dinucleotide (NAD+, an essential coenzyme in ATP production and the sole substrate of the nuclear enzyme poly-ADP-ribose polymerase-1 (PARP-1. Numerous in vitro and in vivo studies have clearly shown that PARP-1 and NAD+ status influence cellular responses to genotoxicity which can lead to mutagenesis and cancer formation. This paper will examine the role of nicotinamide in the protection from carcinogenesis, DNA repair, and maintenance of genomic stability.

  8. Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

    DEFF Research Database (Denmark)

    Beli, Petra; Lukashchuk, Natalia; Wagner, Sebastian A

    2012-01-01

    The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and ...... cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair....

  9. DNA binding, DNA cleavage, and cytotoxicity studies of two new copper (II) complexes.

    Science.gov (United States)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Rezvani, Alireza; Mansouri, Ghobad

    2011-05-01

    The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5 × 10(5) and 5 × 10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.

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

    Science.gov (United States)

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

    1995-12-01

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

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

    Science.gov (United States)

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

    2011-06-03

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

  12. Mechanisms and clinical correlates of sperm DNA damage

    Institute of Scientific and Technical Information of China (English)

    Lara Tamburrino; Sara Marchiani; Margarita Montoya; Francesco Elia Marino; Ilaria Natali; Marta Cambi; Gianni Forti; Elisabetta Baldi; Monica Muratori

    2012-01-01

    Among the different DNA anomalies that can be present in the male gamete,DNA fragmentation is the most frequent,particularly in infertile subjects.There is now consistent evidence that a sperm containing fragmented DNA can be alive,motile,morphologically normal and able to fertilize an oocyte.There is also evidence that the oocyte is able to repair DNA damage; however,the extent of this repair depends on the type of DNA damage present in the sperm,as well as on the quality of the oocyte.Thus,it is important to understand the possible consequences of sperm DNA fragmentation (SDF) for embryo development,implantation,pregnancy outcome and the health of progeny conceived,both naturally and by assisted reproductive technology (ART).At present,data on the consequences of SDF for reproduction are scarce and,in many ways,inconsistent.The differences in study conclusions might result from the different methods used to detect SDF,the study design and the inclusion criteria.Consequently,it is difficult to decide whether SDF testing should be carried out in fertility assessment and ART.It is clear that there is an urgent need for the standardisation of the methods and for additional clinical studies on the impact of SDF on ART outcomes.

  13. Endogenous melatonin and oxidatively damaged guanine in DNA

    Directory of Open Access Journals (Sweden)

    Poulsen Henrik E

    2009-10-01

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

  14. Nanoparticles can cause DNA damage across a cellular barrier

    Science.gov (United States)

    Bhabra, Gevdeep; Sood, Aman; Fisher, Brenton; Cartwright, Laura; Saunders, Margaret; Evans, William Howard; Surprenant, Annmarie; Lopez-Castejon, Gloria; Mann, Stephen; Davis, Sean A.; Hails, Lauren A.; Ingham, Eileen; Verkade, Paul; Lane, Jon; Heesom, Kate; Newson, Roger; Case, Charles Patrick

    2009-12-01

    The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

  15. New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...

  16. The role of the adenovirus DNA binding protein in DNA replication and recombination

    NARCIS (Netherlands)

    Breukelen, B. van

    2003-01-01

    Replication of adenovirus DNA in infected cells is an efficient process that, compared to cellular replication, has the use of a protein primer as a hallmark. The mechanism of this DNA replication process and especially the role of one of the replication proteins, the DNA binding protein DBP, is the

  17. Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

    Science.gov (United States)

    Frégeau, Chantal J; De Moors, Anick

    2012-09-01

    The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields.

  18. Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

    Science.gov (United States)

    Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

    2013-11-01

    A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA

  19. Binding and Transformation of Extracellular DNA in Soil

    Institute of Scientific and Technical Information of China (English)

    CAI Peng; HUANG Qiao-Yun; ZHANG Xue-Wen; CHEN Hao

    2005-01-01

    DNA is the genetic material of various organisms. Extracellular DNA adsorbed or bound on surface-active particles in soils has been shown to persist for long periods against nucleases degradation and still retain the ability to transform competent cells. This paper reviews some recent advances on the binding and transformation of extracellular DNA in soils,which is fundamental to understanding the nature of the soil, regulating biodiversity, and assessing the risk of releasing genetically engineered microorganisms (GEMs) as well as being helpful for development of the genetic evolutional theory of bacteria. Several influencing factors, such as soil pH, ionic strength, soil surface properties, and characteristics of the DNA polymer, are discussed. To date, the understanding of the type of molecular binding sites and the conformation of adsorbed and bound DNA to soil particles is still in its infancy.

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

    OpenAIRE

    Vives, Sergi; Gilbert, Thomas; Arenas, Conchita; Gigli, Elena, 1978-; Lao Grueso, Oscar; Lalueza-Fox, Carles

    2008-01-01

    textabstractBackground. We have analysed the distribution of post mortem DNA damage derived miscoding lesions from the datasets of seven published Neandertal specimens that have extensive cloned sequence coverage over the mitochondrial DNA (mtDNA) hypervariable region 1 (HVS1). The analysis was restricted to C → T and G → A miscoding lesions (the predominant manifestation of post mortem damage) that are seen at a frequency of more than one clone among sequences from a single PCR, but do not r...

  1. Dynamics of nucleosome invasion by DNA binding proteins.

    Science.gov (United States)

    Tims, Hannah S; Gurunathan, Kaushik; Levitus, Marcia; Widom, Jonathan

    2011-08-12

    Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.

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

    Directory of Open Access Journals (Sweden)

    Robert L. Eoff

    2010-01-01

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

  3. Reduction in oxidatively generated DNA damage following smoking cessation

    Directory of Open Access Journals (Sweden)

    Freund Harold G

    2011-05-01

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

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

    Science.gov (United States)

    Fayzullina, Saniya; Martin, Lee J

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Saniya Fayzullina

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

  6. Chromatin compaction protects genomic DNA from radiation damage.

    Directory of Open Access Journals (Sweden)

    Hideaki Takata

    Full Text Available Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs in compact chromatin after ionizing irradiation was 5-50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.

  7. DNA damage due to perfluorooctane sulfonate based on nano-gold embedded in nano-porous poly-pyrrole film

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Liping, E-mail: lipinglu@bjut.edu.cn; Xu, Laihui; Kang, Tianfang; Cheng, Shuiyuan

    2013-11-01

    DNA damage induced from perfluorooctane sulfonate (PFOS) was further developed on a nano-porous bionic interface. The interface was formed by assembling DNA on nano-gold particles which were embedded in a nano-porous overoxidized polypyrrole film (OPPy). Atomic force microscopy, scanning electron microscope and electrochemical investigations indicate that OPPy can be treated to form nano-pore structures. DNA damage due to PFOS was proved using electrochemistry and X-ray photoelectron spectroscopy (XPS) and was investigated by detecting differential pulse voltammetry (DPV) response of methylene blue (MB) which was used as electro-active indicator in the system. The current of MB attenuates obviously after incubation of DNA in PFOS. Moreover, electrochemical impedance spectroscopy (EIS) demonstrates that PFOS weakens DNA charge transport. The tentative binding ratio of PFOS: DNA base pair was obtained by analyzing XPS data of this system.

  8. Characterization of a novel DNA glycosylase from S. sahachiroi involved in the reduction and repair of azinomycin B induced DNA damage.

    Science.gov (United States)

    Wang, Shan; Liu, Kai; Xiao, Le; Yang, LiYuan; Li, Hong; Zhang, FeiXue; Lei, Lei; Li, ShengQing; Feng, Xu; Li, AiYing; He, Jing

    2016-01-01

    Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein-DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    ABSTRACT: BACKGROUND: We have analysed the distribution of post mortem DNA damage derived miscoding lesions from the datasets of seven published Neandertal specimens that have extensive cloned sequence coverage over the mitochondrial DNA (mtDNA) hypervariable region 1 (HVS1). The analysis...... was restricted to C-->T and G-->A miscoding lesions (the predominant manifestation of post mortem damage) that are seen at a frequency of more than one clone among sequences from a single PCR, but do not represent the true endogenous sequence. FINDINGS: The data indicates an extreme bias towards C-->T over G......-->A miscoding lesions (observed ratio of 67:2 compared to an expected ratio of 7:2), implying that the mtDNA Light strand molecule suffers proportionally more damage-derived miscoding lesions than the Heavy strand. CONCLUSION: The clustering of Cs in the Light strand as opposed to the singleton pattern of Cs...

  10. DnaT is a PriC-binding protein.

    Science.gov (United States)

    Huang, Chien-Chih; Huang, Cheng-Yang

    2016-09-01

    DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT-PriC changed from red to purple, which indicated the protein-protein interactions due to gold nanoparticle aggregate. In addition, this DnaT-PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10(-8) M. These results constitute a pioneering study of the DnaT-PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC-SSB-DNA tricomplex and regulates the PriC-dependent replication restart.

  11. enDNA-Prot: Identification of DNA-Binding Proteins by Applying Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Ruifeng Xu

    2014-01-01

    Full Text Available DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97–9.52% in ACC and 0.08–0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83–16.63% in terms of ACC and 0.02–0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public.

  12. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    Science.gov (United States)

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  13. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    Directory of Open Access Journals (Sweden)

    Joerg Kotzerke

    Full Text Available (99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs or double-strand breaks (DSBs; the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99mTc-labeled HYNIC-DAPI compound with that of (99mTc pertechnetate ((99mTcO4(-. pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03 was twice that caused by (99mTcO4(- (0.51, and the number of DSBs increased fivefold in the (99mTc-HYNIC-DAPI-treated sample compared with the (99mTcO4(- treated sample (0.02 to 0.10. In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99mTcO4(- treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99mTc-HYNIC-DAPI-treated samples. These results indicated that (99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99mTc-labeled compound with DNA. In contrast to these results, (99mTcO4(- induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately

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

    Science.gov (United States)

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

    2017-03-01

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

  15. Protection of cadmium chloride induced DNA damage by Lamiaceae plants

    Institute of Scientific and Technical Information of China (English)

    Ramaraj Thirugnanasampandan; Rajarajeswaran Jayakumar

    2011-01-01

    Objective: To analyze the total phenolic content, DNA protecting and radical scavenging activity of ethanolic leaf extracts of three Lamiaceae plants, i.e. Anisomelos malabarica (A. malabarica), Leucas aspera (L. aspera) and Ocimum basilicum (O. basilicum). Methods: The total polyphenols and flavonoids were analyzed in the ethanolic leaf extracts of the lamiaceae plants. To determine the DNA protecting activity, various concentrations of the plant extracts were prepared and treated on cultured HepG2 human lung cancer cells. The pretreated cells were exposed to H2O2 to induce DNA damage through oxidative stress. Comet assay was done and the tail length of individual comets was measured. Nitric oxide and superoxide anion scavenging activities of lamiaceae plants were analyzed. Results: Among the three plant extracts, the highest amount of total phenolic content was found in O. basilicum (189.33 mg/g), whereas A. malabarica showed high levels of flavonoids (10.66 mg/g). O. basilicum also showed high levels of DNA protecting (85%) and radical scavenging activity. Conclusions: The results of this study shows that bioactive phenols present in lamiaceae plants may prevent carcinogenesis through scavenging free radicals and inhibiting DNA damage.

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

    Science.gov (United States)

    Hayashi, Michio; Umezu, Keiko

    2017-04-03

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

  17. X-Ray induced DNA damage – why use plants?

    Directory of Open Access Journals (Sweden)

    John William Einset

    2015-06-01

    Full Text Available The comet assay was used to monitor DNA repair after X-ray exposures caused by 0.2-15 Gy. A clear distinction in the time course of DNA repair after 2 Gy was observed with an early ‘rapid phase’, lasting 20-40 minutes, being followed by a ‘slow phase’ which actually consists of a period of negligible repair and then rapid repair during 140-160 minutes. The fact that homozygous mutants for both ATM and BRCA1 fail to repair DNA completely during 3 hours after 2 Gy exposures indicates that repair processes occurring during the ‘slow phase’ involve ds breaks in DNA. Both BRCA1 and Rad51 expression are strongly upregulated by X-rays in Arabidopsis. Rye grass, Norway spruce and Sawara cypress also have ‘slow phase’ repair similar to Arabidopsis, suggesting that the requisite enzymes have to be induced in these plants as well. To look at the effect of genome size in relation to sensitivity to DNA damage, we exposed isolated nuclei from Norway spruce (19.2 Gbp genome, celery (14.1 Gbp, spinach (12.6 Gbp Sawara cypress (8.9 Gbp, lettuce (2.6 Gbp and Arabidopsis (0.135 Gbp to X-rays. After a 1 Gy exposure, a linear relationship was seen between % tails and genome size, confirming the idea that larger genomes are more sensitive to X-ray damage.

  18. Decreased repair of gamma damaged DNA in progeria

    Energy Technology Data Exchange (ETDEWEB)

    Rainbow, A.J.; Howes, M.

    1977-01-01

    A sensitive host-cell reactivation technique was used to examine the DNA repair ability of fibroblasts from two patients with classical progeria. Fibroblasts were infected with either non-irradiated or gamma-irradiated adenovirus type 2 and at 48 hrs after infection cells were examined for the presence of viral structural antigens using immunofluorescent staining. The production of viral structural antigens was considerably reduced in the progeria lines as compared to normal fibroblasts when gamma-irradiated virus was used, indicating a defect in the repair of gamma ray damaged DNA in the progeria cells.

  19. 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...... and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10-5), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using...

  20. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    N R Jena

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA–protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA–protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.

  1. Preservation of ancient DNA in thermally damaged archaeological bone

    Science.gov (United States)

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

    2009-02-01

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

  2. PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

    Science.gov (United States)

    Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

    2013-06-14

    Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

  3. Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

    Energy Technology Data Exchange (ETDEWEB)

    Nandi, Chayan K.; Parui, Partha P.; Brutschy, Bernhard [University of Frankfurt, Institute for Physical and Theoretical Chemistry, Frankfurt (Germany); Schmidt, Thorsten L.; Heckel, Alexander [University of Frankfurt, Cluster of Excellence Macromolecular Complexes, c/o Institute for Organic Chemistry and Chemical Biology, Frankfurt (Germany)

    2008-03-15

    We have recently constructed a ''DNA strut'' consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson-Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions. (orig.)

  4. Personal exposure to ultrafine particles and oxidative DNA damage

    DEFF Research Database (Denmark)

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

    2005-01-01

    10), nitrous oxide, nitrogen dioxide, carbon monoxide, and/or number concentration of UFPs at urban background or busy street monitoring stations was not a significant predictor of DNA damage, although personal UFP exposure was correlated with urban background concentrations of CO and NO2......Exposure to ultrafine particles (UFPs) from vehicle exhaust has been related to risk of cardiovascular and pulmonary disease and cancer, even though exposure assessment is difficult. We studied personal exposure in terms of number concentrations of UFPs in the breathing zone, using portable...... instruments in six 18-hr periods in 15 healthy nonsmoking subjects. Exposure contrasts of outdoor pollution were achieved by bicycling in traffic for 5 days and in the laboratory for 1 day. Oxidative DNA damage was assessed as strand breaks and oxidized purines in mononuclear cells isolated from venous blood...

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

    DEFF Research Database (Denmark)

    Jørgensen, Anders

    2013-01-01

    -oxodG and 8-oxoGuo, respectively). The main hypothesis was that psychological stress states are associated with increased DNA/RNA damage from oxidation. In a study of 40 schizophrenia patients and 40 healthy controls matched for age and gender, we found that 8-oxodG/8-oxoGuo excretion was increased...... correlations between 8-oxodG/8-ocoGuo excretion and 9AM plasma cortisol, but no associations to perceived stress. In an animal study of experimentally induced chronic stress performed in metabolism cages, we found no increase in urinary 8-oxodG/8-oxoGuo or cerebral (hippocampal and frontal cortex) levels...... between the 24 h urinary cortisol excretion and the excretion of 8-oxodG/8-oxoGuo, determined in the same samples. Collectively, the studies could not confirm an association between psychological stress and oxidative stress on nucleic acids. Systemic oxidatively generated DNA/RNA damage was increased...

  6. 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.

  7. Solution structure and binding specificity of the p63 DNA binding domain.

    Science.gov (United States)

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-05-26

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner.

  8. Pyrosequencing: applicability for studying DNA damage-induced mutagenesis.

    Science.gov (United States)

    Minko, Irina G; Earley, Lauriel F; Larlee, Kimberly E; Lin, Ying-Chih; Lloyd, R Stephen

    2014-10-01

    Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre-determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N(6) -(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5'-GCNGG-3' sequence context. Pyrosequencing detected ∼8% G to T transversions and ∼3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108-1114). However, ∼3.5% G to C transversions and ∼2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ∼1-2% for A and T and ∼5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-01-18

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

  10. R-Roscovitine (Seliciclib) prevents DNA damage-induced cyclin A1 upregulation and hinders non-homologous end-joining (NHEJ) DNA repair.

    Science.gov (United States)

    Federico, Mario; Symonds, Catherine E; Bagella, Luigi; Rizzolio, Flavio; Fanale, Daniele; Russo, Antonio; Giordano, Antonio

    2010-08-04

    CDK-inhibitors can diminish transcriptional levels of cell cycle-related cyclins through the inhibition of E2F family members and CDK7 and 9. Cyclin A1, an E2F-independent cyclin, is strongly upregulated under genotoxic conditions and functionally was shown to increase NHEJ activity. Cyclin A1 outcompetes with cyclin A2 for CDK2 binding, possibly redirecting its activity towards DNA repair. To see if we could therapeutically block this switch, we analyzed the effects of the CDK-inhibitor R-Roscovitine on the expression levels of cyclin A1 under genotoxic stress and observed subsequent DNA damage and repair mechanisms. We found that R-Roscovitine alone was unable to alter cyclin A1 transcriptional levels, however it was able to reduce protein expression through a proteosome-dependent mechanism. When combined with DNA damaging agents, R-Roscovitine was able to prevent the DNA damage-induced upregulation of cyclin A1 on a transcriptional and post-transcriptional level. This, moreover resulted in a significant decrease in non-homologous end-joining (NHEJ) paired with an increase in DNA DSBs and overall DNA damage over time. Furthermore, microarray analysis demonstrated that R-Roscovitine affected DNA repair mechanisms in a more global fashion. Our data reveal a new mechanism of action for R-Roscovitine on DNA repair through the inhibition of the molecular switch between cyclin A family members under genotoxic conditions resulting in reduced NHEJ capability.

  11. Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

    Science.gov (United States)

    Eide, L; Bjørås, M; Pirovano, M; Alseth, I; Berdal, K G; Seeberg, E

    1996-10-01

    One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

  12. 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.

  13. 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.

  14. The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.

    Science.gov (United States)

    Takada, Saeko; Collins, Eric R; Kurahashi, Kayo

    2015-05-15

    DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.

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

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

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

  16. ATM kinase: Much more than a DNA damage responsive protein.

    Science.gov (United States)

    Guleria, Ayushi; Chandna, Sudhir

    2016-03-01

    ATM, mutation of which causes Ataxia telangiectasia, has emerged as a cardinal multifunctional protein kinase during past two decades as evidenced by various studies from around the globe. Further to its well established and predominant role in DNA damage response, ATM has also been understood to help in maintaining overall functional integrity of cells; since its mutation, inactivation or deficiency results in a variety of pathological manifestations besides DNA damage. These include oxidative stress, metabolic syndrome, mitochondrial dysfunction as well as neurodegeneration. Recently, high throughput screening using proteomics, metabolomics and transcriptomic studies revealed several proteins which might be acting as substrates of ATM. Studies that can help in identifying effective regulatory controls within the ATM-mediated pathways/mechanisms can help in developing better therapeutics. In fact, more in-depth understanding of ATM-dependent cellular signals could also help in the treatment of variety of other disease conditions since these pathways seem to control many critical cellular functions. In this review, we have attempted to put together a detailed yet lucid picture of the present-day understanding of ATM's role in various pathophysiological conditions involving DNA damage and beyond.

  17. Oxidative DNA damage in mouse sperm chromosomes: Size matters.

    Science.gov (United States)

    Kocer, Ayhan; Henry-Berger, Joelle; Noblanc, Anais; Champroux, Alexandre; Pogorelcnik, Romain; Guiton, Rachel; Janny, Laurent; Pons-Rejraji, Hanae; Saez, Fabrice; Johnson, Graham D; Krawetz, Stephen A; Alvarez, Juan G; Aitken, R John; Drevet, Joël R

    2015-12-01

    Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization.

  18. Microvesicles Contribute to the Bystander Effect of DNA Damage.

    Science.gov (United States)

    Lin, Xiaozeng; Wei, Fengxiang; Major, Pierre; Al-Nedawi, Khalid; Al Saleh, Hassan A; Tang, Damu

    2017-04-07

    Genotoxic treatments elicit DNA damage response (DDR) not only in cells that are directly exposed but also in cells that are not in the field of treatment (bystander cells), a phenomenon that is commonly referred to as the bystander effect (BE). However, mechanisms underlying the BE remain elusive. We report here that etoposide and ultraviolet (UV) exposure stimulate the production of microvesicles (MVs) in DU145 prostate cancer cells. MVs isolated from UV-treated DU145 and A431 epidermoid carcinoma cells as well as etoposide-treated DU145 cells induced phosphorylation of ataxia-telangiectasia mutated (ATM) at serine 1981 (indicative of ATM activation) and phosphorylation of histone H2AX at serine 139 (γH2AX) in naïve DU145 cells. Importantly, neutralization of MVs derived from UV-treated cells with annexin V significantly reduced the MV-associated BE activities. Etoposide and UV are known to induce DDR primarily through the ATM and ATM- and Rad3-related (ATR) pathways, respectively. In this regard, MV is likely a common source for the DNA damage-induced bystander effect. However, pre-treatment of DU145 naïve cells with an ATM (KU55933) inhibitor does not affect the BE elicited by MVs isolated from etoposide-treated cells, indicating that the BE is induced upstream of ATM actions. Taken together, we provide evidence supporting that MVs are a source of the DNA damage-induced bystander effect.

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

    Science.gov (United States)

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

    2016-01-01

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

  20. CLK-1 protein has DNA binding activity specific to O(L) region of mitochondrial DNA.

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei

    2002-04-10

    Mutations in the clk-1 gene of Caenorhabditis elegans extend worm life span and slow down a variety of physiological processes. Here we report that C. elegans CLK-1 as well as its mouse homologue have DNA binding activity that is specific to the O(L) region of mitochondrial DNA. DNA binding activity of CLK-1 is inhibited by ADP, and is altered by mutations that extend nematode life span. Our results suggest that, in addition to its enzymatic function in ubiquinone biosynthesis, CLK-1 is involved in the regulation of mtDNA replication or transcription.

  1. Synthesis, DNA binding and cytotoxic evaluation of aminoquinoline scaffolds

    Indian Academy of Sciences (India)

    Gopal Senthil Kumar; Mohamed Ashraf Ali; Tan Soo Choon; Rajendra Prasad Karnam Jayarampillai

    2016-03-01

    An effortless synthetic route has been developed for the synthesis of a new class of aminoquinoline substituted isoindolin-1,3-diones from regio-isomerical hydrazinylquinolines with phthalic anhydride in presence of Eaton’s reagent. DNA binding studies of selected isomeric compounds showed interaction withDNA via intercalation mode with higher binding affinity of 4-substituted quinolines rather than 2-substituted counterparts. Further, all compounds were screened for cytotoxic activity against three human cancer cell lines,among them compound 2c outranged standard doxorubicin against CCRF-CEM cell line.

  2. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  3. DNA damage in workers occupationally exposed to pesticide mixtures.

    Science.gov (United States)

    Simoniello, M F; Kleinsorge, E C; Scagnetti, J A; Grigolato, R A; Poletta, G L; Carballo, M A

    2008-11-01

    Pesticides are used in agriculture to protect crops but represent at the same time a potential risk to farmers and environment. The aim of this work is the evaluation of 54 subjects occupationally exposed to pesticides and 30 subjects as a control group using the quantification of DNA damage level by means of the alkaline Comet assay and the evaluation of repair processes. Damage index Comet assay (DICA) and damage index repair assay (DIRA) were studied in 27 pesticide applicator workers, 27 non-pesticide applicators and controls. Our results show that both exposed groups revealed significant increase in DICA when compared with controls (P pesticides was investigated and no significant differences were observed considering age, gender, smoking and alcohol consumption in relation to DICA and DIRA. Since DNA damage is an important step in events leading from carcinogen exposure to cancer disease, our study highlights the potential health risk associated with agrochemical exposure in developing countries with vast cultivated areas, such as Argentina.

  4. Giardia duodenalis Rad52 protein: biochemical characterization and response upon DNA damage.

    Science.gov (United States)

    Martínez-Miguel, Rosa María; Sandoval-Cabrera, Antonio; Bazán-Tejeda, María Luisa; Torres-Huerta, Ana Laura; Martínez-Reyes, Diego A; Bermúdez-Cruz, Rosa María

    2017-08-01

    Giardia duodenalis is a flagellated binucleated protozoan that colonizes the small intestine in mammals, causing giardiasis, acute or chronic diarrhea. DNA double strand break either endogenously or exogenously generated is a major insult to DNA and its repair by homologous recombination (HR) is crucial for genomic stability. During HR, Rad52 plays key roles in the loading of the Rad51 recombinase, and the annealing of the second double-strand break end to the displaced strand of the D-loop structure. Among the functions found in vitro in yeast and human Rad52 protein are: ssDNA or dsDNA binding activity, ability to anneal bare or RPA coated-ssDNA, as well as multimeric ring formation. In this work, we searched for conserved domains in a putative Rad52 protein from G. duodenalis (GdRad52). Its coding sequence was cloned, expressed and purified to study its biochemical properties. rGdRad52 binds to dsDNA and ssDNA, with greater affinity for the latter. Likewise, rGdRad52 promotes annealing of DNA uncoated and coated with GdRPA1. rGdRad52 interacts with GdDMC1B and with GdRPA1 protein as shown in far western blotting assay. Additionally, rGdRad52 formed multimeric rings as observed by electronic microscopy. Finally, GdRad52 is over expressed in response upon DNA damage inflicted on trophozoites. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  5. Cryo-EM Imaging of DNA-PK DNA Damage Repair Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Phoebe L. Stewart

    2005-06-27

    Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second

  6. Environmental car exhaust pollution damages human sperm chromatin and DNA.

    Science.gov (United States)

    Calogero, A E; La Vignera, S; Condorelli, R A; Perdichizzi, A; Valenti, D; Asero, P; Carbone, U; Boggia, B; De Rosa, N; Lombardi, G; D'Agata, R; Vicari, L O; Vicari, E; De Rosa, M

    2011-06-01

    The adverse role of traffic pollutants on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled. All of them were interviewed about their lifestyle. Hormone, semen samples, and environmental and biological markers of pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. LH, FSH, and testosterone serum levels were within the normal range in tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were significantly lower in these men compared with controls. Motorway tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a late sign of apoptosis, compared with controls. A significant direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship. This study showed that car exhaust exposure has a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance of the men exposed, but also for the offspring health.

  7. Preparation of next-generation sequencing libraries from damaged DNA.

    Science.gov (United States)

    Briggs, Adrian W; Heyn, Patricia

    2012-01-01

    Next-generation sequencing (NGS) has revolutionized ancient DNA research, especially when combined with high-throughput target enrichment methods. However, attaining high sequencing depth and accuracy from samples often remains problematic due to the damaged state of ancient DNA, in particular the extremely low copy number of ancient DNA and the abundance of uracil residues derived from cytosine deamination that lead to miscoding errors. It is therefore critical to use a highly efficient procedure for conversion of a raw DNA extract into an adaptor-ligated sequencing library, and equally important to reduce errors from uracil residues. We present a protocol for NGS library preparation that allows highly efficient conversion of DNA fragments into an adaptor-ligated form. The protocol incorporates an option to remove the vast majority of uracil miscoding lesions as part of the library preparation process. The procedure requires only two spin column purification steps and no gel purification or bead handling. Starting from an aliquot of DNA extract, a finished, highly amplified library can be generated in 5 h, or under 3 h if uracil removal is not required.

  8. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    Science.gov (United States)

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  9. LNA effects on DNA binding and conformation

    DEFF Research Database (Denmark)

    Pabon-Martinez, Y Vladimir; Xu, You; Villa, Alessandra

    2017-01-01

    hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked...... nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3'-end of a TFO destabilizes the triplex...... structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3'-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA...

  10. DNA-damage accumulation and replicative arrest in Hutchinson–Gilford progeria syndrome

    Science.gov (United States)

    Musich, Phillip R.; Zou, Yue

    2013-01-01

    A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson–Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression. PMID:22103522

  11. DNA-damage accumulation and replicative arrest in Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Musich, Phillip R; Zou, Yue

    2011-12-01

    A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

  12. Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage.

    Science.gov (United States)

    Pereg, Yaron; Lam, Suzanne; Teunisse, Amina; Biton, Sharon; Meulmeester, Erik; Mittelman, Leonid; Buscemi, Giacomo; Okamoto, Koji; Taya, Yoichi; Shiloh, Yosef; Jochemsen, Aart G

    2006-09-01

    The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

  13. Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

    Science.gov (United States)

    Kang, Ho Chul; Lee, Yun-Il; Shin, Joo-Ho; Andrabi, Shaida A.; Chi, Zhikai; Gagné, Jean-Philippe; Lee, Yunjong; Ko, Han Seok; Lee, Byoung Dae; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair. PMID:21825151

  14. Effect of knots on binding of intercalators to DNA

    Science.gov (United States)

    Medalion, Shlomi; Rabin, Yitzhak

    2014-05-01

    We study the effect of knots in circular dsDNA molecules on the binding of intercalating ligands. Using Monte Carlo simulations we show that depending on their handedness, the presence of knots can either suppress or enhance intercalation in supercoiled DNA. When the occupancy of intercalators on DNA is low, the effect of knots on intercalation can be captured by introducing a shift in the mean writhe of the chain that accounts for the writhe of the corresponding ideal knot. In the limit of high intercalator occupancy, the writhe distribution of different knots is strongly affected by excluded volume effects and therefore by salt concentration. Based on the finding that different knots yield well-separated probability distributions of bound intercalators, we propose a new experimental approach to determine DNA topology by monitoring the intensity of fluorescence emitted by dye molecules intercalated into knotted DNA molecules.

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

    NARCIS (Netherlands)

    Yi, Xia

    2007-01-01

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

  16. Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

    Science.gov (United States)

    Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M; Hiasa, Hiroshi; Marks, Kevin R; Kerns, Robert J; Berger, James M; Drlica, Karl

    2014-05-02

    DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

  17. Mitochondrial DNA damage and animal longevity: insights from comparative studies.

    Science.gov (United States)

    Pamplona, Reinald

    2011-03-02

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

  18. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan;

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by...

  19. Evidence for DNA Damage as a Biological Link Between Diabetes and Cancer

    Institute of Scientific and Technical Information of China (English)

    Shao Chin Lee; Juliana CN Chan

    2015-01-01

    Objective:This review examines the evidence that:Diabetes is a state of DNA damage;pathophysiological factors in diabetes can cause DNA damage;DNA damage can cause mutations;and DNA mutation is linked to carcinogenesis.Data Sources:We retrieved information from the PubMed database up to January,2014,using various search terms and their combinations including DNA damage,diabetes,cancer,high glucose,hyperglycemia,free fatty acids,palmitic acid,advanced glycation end products,mutation and carcinogenesis.Study Selection:We included data from peer-reviewed journals and a textbook printed in English on relationships between DNA damage and diabetes as well as pathophysiological factors in diabetes.Publications on relationships among DNA damage,mutagenesis,and carcinogenesis,were also reviewed.We organized this information into a conceptual framework to explain the possible causal relationship between DNA damage and carcinogenesis in diabetes.Results:There are a large amount of data supporting the view that DNA mutation is a typical feature in carcinogenesis.Patients with type 2 diabetes have increased production of reactive oxygen species,reduced levels of antioxidant capacity,and increased levels of DNA damage.The pathophysiological factors and metabolic milieu in diabetes can cause DNA damage such as DNA strand break and base modification (i.e.,oxidation).Emerging experimental data suggest that signal pathways (i.e.,Akt/tuberin) link diabetes to DNA damage.This collective evidence indicates that diabetes is a pathophysiological state of oxidative stress and DNA damage which can lead to various types of mutation to cause aberration in cells and thereby increased cancer risk.Conclusions:This review highlights the interrelationships amongst diabetes,DNA damage,DNA mutation and carcinogenesis,which suggests that DNA damage can be a biological link between diabetes and cancer.

  20. MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Christopher J. Staples

    2016-09-01

    Full Text Available Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP. We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP’s role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response.

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

    Science.gov (United States)

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

    2016-11-01

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

  2. Extended HSR/CARD domain mediates AIRE binding to DNA.

    Science.gov (United States)

    Maslovskaja, Julia; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-12-25

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Effects of DNA damage on oocyte meiotic maturation and early embryonic development

    Directory of Open Access Journals (Sweden)

    Shen YIN,Junyu MA,Wei SHEN

    2014-09-01

    Full Text Available DNA damage is one of the most common threats to meiotic cells. It has the potential to induce infertility and genetic abnormalities that may be passed to the embryo. Here, we reviewed exogenous factors which could induce DNA damage. Specially, we addressed the different effects of DNA damage on mouse oocytes and embryonic development. Complex DNA damage, double-strand breaks, represents a more difficult repair process and involves various repair pathways. Understanding the mechanisms involved in DNA damage responses may improve therapeutic strategies for ovarian cancer and fertility preservation.

  4. Studies on DNA Damage Response in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Han, Wenyuan

    global reactions known as DNA damage response (DDR). In Bacteria and Eukaryotes, the global reactions include a series of transcription regulations and protein post-translation modifications, which can activate DNA repair machineries, suppress cell division and delay DNA replication, and induce...... scattered light, damaged cell membrane and electron-dense area. During NQO and MMS treatment, degradation of chromatin proteins was coincided with DNA-less cell formation, suggesting their roles in protecting genomic DNA from massive degradation. Further, HU inhibited NQO-induced DSB formation and DNA...... damage response, suggesting the crucial roles of DSB in triggering DNA damage response. Then, NQO-induced DNA-less formation was impaired in the culture with retarded cell cycle, suggesting that DNA replication played an important role in DNA damage response in Sulfolobus. We also investigated the roles...

  5. Sperm DNA damage in men from infertile couples

    Institute of Scientific and Technical Information of China (English)

    Juris Erenpreiss; Saad Elzanaty; Aleksander Giwercman

    2008-01-01

    Aim: To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. Methods: A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay (SCSA) were carried out. Results: Ninety-seven men (28% of the whole study group) had a DNA fragmentation index (DFI) > 20%, and 43 men (12%) had a DFI > 30%. In the group of men with abnormal semen parameters (n = 224), 35% had a DFI > 20%, and 16% had a DFI > 30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters (n = 126). Men with low sperm motility and abnormal morphology had significantly higher odds ratios (Ors) for having a DFI > 20% (4.0 for motility and 1.9 for morphology) and DFI > 30% (6.2 for motility and 2.8 for morphology) compared with men with normal sperm motility and morphology. Conclusion: In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique (ART).

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

    Science.gov (United States)

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

    2014-01-01

    There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate–activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism. PMID:24395638

  7. DNA damage and mutations induced by arachidonic acid peroxidation.

    Science.gov (United States)

    Lim, Punnajit; Sadre-Bazzaz, Kianoush; Shurter, Jesse; Sarasin, Alain; Termini, John

    2003-12-30

    Endogenous cellular oxidation of omega6-polyunsaturated fatty acids (PUFAs) has long been recognized as a contributing factor in the development of various cancers. The accrual of DNA damage as a result of reaction with free radical and electrophilic aldehyde products of lipid peroxidation is believed to be involved; however, the genotoxic and mutation-inducing potential of specific membrane PUFAs remains poorly defined. In the present study we have examined the ability of peroxidizing arachidonic acid (AA, 20:4omega6) to induce DNA strand breaks, base modifications, and mutations. The time-dependent induction of single-strand breaks and oxidative base modifications by AA in genomic DNA was quantified using denaturing glyoxal gel electrophoresis. Mutation spectra were determined in XP-G fibroblasts and a repair-proficient line corrected for this defect by c-DNA complementation (XP-G(+)). Mutation frequencies were elevated from approximately 5- to 30-fold over the background following reaction of DNA with AA for various times. The XPG gene product was found to be involved in the suppression of mutations after extended reaction of DNA with AA. Arachidonic acid-induced base substitutions were consistent with the presence of both oxidized and aldehyde base adducts in DNA. The frequency of multiple-base substitutions induced by AA was significantly reduced upon correction for the XPG defect (14% vs 2%, P = 0.0015). Evidence is also presented which suggests that the induced frequency of multiple mutations is lesion dependent. These results are compared to published data for mutations stimulated by alpha,beta-unsaturated aldehydes identified as products of lipid peroxidation.

  8. Selection against spermatozoa with fragmented DNA after postovulatory mating depends on the type of damage

    Directory of Open Access Journals (Sweden)

    Pintado Belén

    2010-01-01

    Full Text Available Abstract Background Before ovulation, sperm-oviduct interaction mechanisms may act as checkpoint for the selection of fertilizing spermatozoa in mammals. Postovulatory mating does not allow the sperm to attach to the oviduct, and spermatozoa may only undergo some selection processes during the transport through the female reproductive tract and/or during the zona pellucida (ZP binding/penetration. Methods We have induced DNA damage in spermatozoa by two treatments, (a a scrotal heat treatment (42 degrees C, 30 min and (b irradiation with 137Cs gamma-rays (4 Gy, 1.25 Gy/min. The effects of the treatments were analyzed 21-25 days post heat stress or gamma-radiation. Postovulatory females mated either with treated or control males were sacrificed at Day 14 of pregnancy, and numbers of fetuses and resorptions were recorded. Results Both treatments decreased significantly implantation rates however, the proportion of fetuses/resorptions was only reduced in those females mated to males exposed to radiation, indicating a selection favoring fertilization of sperm with unfragmented DNA on the heat treatment group. To determine if DNA integrity is one of the keys of spermatozoa selection after postovulatory mating, we analyzed sperm DNA fragmentation by COMET assay in: a sperm recovered from mouse epididymides; b sperm recovered from three different regions of female uterine horns after mating; and c sperm attached to the ZP after in vitro fertilization (IVF. Similar results were found for control and both treatments, COMET values decreased significantly during the transit from the uterine section close to the uterotubal junction to the oviduct, and in the spermatozoa attached to ZP. However, fertilization by IVF and intracytoplasmatic sperm injection (ICSI showed that during sperm ZP-penetration, a stringent selection against fragmented-DNA sperm is carried out when the damage was induced by heat stress, but not when DNA fragmentation was induced by

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

    Directory of Open Access Journals (Sweden)

    Nan Li

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

  10. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors

    OpenAIRE

    2009-01-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein–DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are esse...

  11. BuD, a helix–loop–helix DNA-binding domain for genome modification

    Energy Technology Data Exchange (ETDEWEB)

    Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

    2014-07-01

    Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

  12. An electrochemical DNA-sensor developed with the use of methylene blue as a redox indicator for the detection of DNA damage induced by endocrine-disrupting compounds

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Xiaoyun [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); College of Chemistry, Nanchang University, Nanchang 330031 (China); Ni, Yongnian, E-mail: ynni@ncu.edu.cn [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); College of Chemistry, Nanchang University, Nanchang 330031 (China); Kokot, Serge, E-mail: s.kokot@qut.edu.au [School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, Brisbane 4001 (Australia)

    2015-03-31

    Highlights: • A new method for detecting DNA damage was successfully developed. • A novel biosensor, MB/dsDNA/GO-CS/AuNPs/GCE biosensor was constructed. • Loading/release of MB in/out of dsDNA/GO-CS/AuNPs film was investigated. • DNA damage induced by BPA, NP and OP was detected and estimated. - Abstract: An electrochemical biosensor capable of indirect detection of DNA damage induced by any one of the three endocrine-disrupting compounds (EDCs) – bisphenol A (BPA), 4-nonylphenol (NP) and 4-t-octylphenol (OP), has been researched and developed. The methylene blue (MB) dye was used as the redox indicator. The glassy carbon electrode (GCE) was modified by the assembled dsDNA/graphene oxide-chitosan/gold nano-particles to produce a dsDNA/GO-CS/AuNPs/GCE sensor. It was characterized with the use of electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and scanning electron microscopy (SEM) techniques. The loading/release of the MB dye by the dsDNA/GO-CS/AuNPs film was investigated, and the results showed that the process was reversible. Based on this, the sensor was used to measure the difference between the loading capabilities of intact and damaged dsDNA in the films. The sensor was then successfully applied to detect DNA damage electrochemically. The differential pulse voltammetry (DPV) peak current ratio for MB, observed before and after DNA damage, increased linearly in the presence the BPA, NP or OP compounds; the treatment range was 10–60 min, and the respective damage rates were 0.0069, 0.0044 and 0.0031 min{sup −1}, respectively. These results were confirmed by the binding constants: 2.09 × 10{sup 6} M{sup −1} (BPA-DNA), 1.28 × 10{sup 6} M{sup −1} (NP-DNA) and 9.33 × 10{sup 5} M{sup −1} (OP-DNA), all of which were obtained with the use of differential pulse stripping voltammetry (DPSV)

  13. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  14. Z-DNA binding protein from chicken blood nuclei

    Science.gov (United States)

    Herbert, A. G.; Spitzner, J. R.; Lowenhaupt, K.; Rich, A.

    1993-01-01

    A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

  15. Recognition rules for binding of homeodomains to operator DNA.

    Science.gov (United States)

    Chirgadze, Yu N; Sivozhelezov, V S; Polozov, R V; Stepanenko, V A; Ivanov, V V

    2012-01-01

    The spatial arrangement of interfaces between homeodomain transcription factors and operator DNA has been considered. We analyzed the binding contacts for a representative set of 22 complexes of homeodomain transcription factors with a double-stranded operator DNA in the region of the major groove. It was shown that the recognition of DNA by the recognizing _-helix of protein is governed by two contact groups. Invariant protein-DNA group of contacts includes six contacts, formed by atomic groups of coding and non-coding DNA chains with the groups of amino acids. The recognizing _-helix forms contacts by polar groups of residues Trp2 (NE1), Asn5, and Lys9 with the canonical sequence T(1)A(2)A(3)T(4) of the coding DNA chain, and contacts by residues Lys0, Arg7 and Lys11 with the sequence A(4)X(5)X(6)X(7) of a non-coding DNA chain, where X is any nucleotide. Variable protein-DNA group of contacts comprises two groups bound with the sequence T(3)A(4)X(5)X(6) of the non- coding DNA-chain. These contacts are mainly with the bases and specify the binding pattern of individual homeodomains. The invariant contact group represents a recognition pattern for transcription factors of the homeodomain family: multiple adenine-asparagine contact and six position-specific phosphate contacts mainly with lysine or arginine. Within this group, we have found three most significant invariant contacts which allow deducing the recognition rules for homeodomains. These rules are inherent for different taxonomic groups of the homeodomain family and can distinguishing members of this family from any other family of transcription factors.

  16. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  17. Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism

    Science.gov (United States)

    Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

    2016-01-01

    Purpose: The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. Methods: The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2- scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays. Results: Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2- scavenging, DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Conclusion: Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3’,4’-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form. PMID:27478791

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

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawłowska

    2017-07-01

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

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

    Science.gov (United States)

    2007-04-01

    Li, 2005). Task 2: Establish whether pre-RC reformation , re-initiation or re-elongation induces the DNA damage response. In task 2 of the...300 l of 0.5-mm glass beads (Biospec Products, Bartlesville, OK) and 300 l of SDS-PAGE loading buffer [8% glycerol (vol/vol), 100 mM Tris-HCl, pH

  20. Polo-like kinase 1 inhibits DNA damage response during mitosis.

    Science.gov (United States)

    Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

    2015-01-01

    In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

  1. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.

    Science.gov (United States)

    Stewart, Grant S; Panier, Stephanie; Townsend, Kelly; Al-Hakim, Abdallah K; Kolas, Nadine K; Miller, Edward S; Nakada, Shinichiro; Ylanko, Jarkko; Olivarius, Signe; Mendez, Megan; Oldreive, Ceri; Wildenhain, Jan; Tagliaferro, Andrea; Pelletier, Laurence; Taubenheim, Nadine; Durandy, Anne; Byrd, Philip J; Stankovic, Tatjana; Taylor, A Malcolm R; Durocher, Daniel

    2009-02-06

    The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.

  2. Lymphocyte DNA damage in Turkish asphalt workers detected by the comet assay.

    Science.gov (United States)

    Bacaksiz, Aysegul; Kayaalti, Zeliha; Soylemez, Esma; Tutkun, Engin; Soylemezoglu, Tulin

    2014-01-01

    Asphalt has a highly complex structure and it contains several organic compounds including polycyclic aromatic hydrocarbons and heterocyclic compounds. In this study, comet assay was used to detect the DNA damage in blood lymphocytes of 30 workers exposed to asphalt fumes and 30 nonexposed controls. This is the first report on Turkish asphalt workers' investigated DNA damage using the alkaline single cell gel electrophoresis (SCGE). The DNA damage was evaluated by the percentage of DNA in the comet tail (% tail DNA) for each cell. According to our results, workers exposed to asphalt fumes had higher DNA damage than the control group (p asphalt fumes caused a significant increase in DNA damage and the comet assay is a suitable method for determining DNA damage in asphalt workers.

  3. Senescence of primary amniotic cells via oxidative DNA damage.

    Directory of Open Access Journals (Sweden)

    Ramkumar Menon

    Full Text Available OBJECTIVE: Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB and preterm prelabor rupture of the membranes (pPROM; however, the precise mechanistic role of reactive oxygen species (ROS in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE, a predicted cause of pregnancy complications, on human amnion epithelial cells. METHODS: Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2'7'-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1, phospho-p38 (P-p38 MAPK, and p19(arf. Expression of senescence-associated β-galectosidase (SAβ-gal was used to confirm cell ageing in situ. RESULTS: ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min and significantly (p<0.01 at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19(Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19(Arf were not significantly affected by NAC. CONCLUSIONS: The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.

  4. Chk2 Activation Dependence on Nbs1 after DNA Damage

    OpenAIRE

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

    2001-01-01

    The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G1 arrest. Here we show that the ATM-dependent activation of Chk2 by γ- radiation requires Nbs1, the gene product ...

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

    Directory of Open Access Journals (Sweden)

    Gigli Elena

    2008-07-01

    Full Text Available Abstract Background We have analysed the distribution of post mortem DNA damage derived miscoding lesions from the datasets of seven published Neandertal specimens that have extensive cloned sequence coverage over the mitochondrial DNA (mtDNA hypervariable region 1 (HVS1. The analysis was restricted to C→T and G→A miscoding lesions (the predominant manifestation of post mortem damage that are seen at a frequency of more than one clone among sequences from a single PCR, but do not represent the true endogenous sequence. Findings The data indicates an extreme bias towards C→T over G→A miscoding lesions (observed ratio of 67:2 compared to an expected ratio of 7:2, implying that the mtDNA Light strand molecule suffers proportionally more damage-derived miscoding lesions than the Heavy strand. Conclusion The clustering of Cs in the Light strand as opposed to the singleton pattern of Cs in the Heavy strand could explain the observed bias, a phenomenon that could be further tested with non-PCR based approaches. The characterization of the HVS1 hotspots will be of use to future Neandertal mtDNA studies, with specific regards to assessing the authenticity of new positions previously unknown to be polymorphic.

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

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

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

  7. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

    Directory of Open Access Journals (Sweden)

    Paul J Holland

    Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  8. Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors.

    Science.gov (United States)

    Gureev, Artem P; Shaforostova, Ekaterina A; Starkov, Anatoly A; Popov, Vasily N

    2017-05-01

    Damage to mitochondrial DNA (mtDNA) is a meaningful biomarker for evaluating genotoxicity of drugs and environmental toxins. Existing PCR methods utilize long mtDNA fragments (∼8-10kb), which complicates detecting exact sites of mtDNA damage. To identify the mtDNA regions most susceptible to damage, we have developed and validated a set of primers to amplify ∼2kb long fragments, while covering over 95% of mouse mtDNA. We have modified the detection method by greatly increasing the enrichment of mtDNA, which allows us solving the problem of non-specific primer annealing to nuclear DNA. To validate our approach, we have determined the most damage-susceptible mtDNA regions in mice treated in vivo and in vitro with rotenone and H2O2. The GTGR-sequence-enriched mtDNA segments located in the D-loop region were found to be especially susceptible to damage. Further, we demonstrate that H2O2-induced mtDNA damage facilitates the relaxation of mtDNA supercoiled conformation, making the sequences with minimal damage more accessible to DNA polymerase, which, in turn, results in a decrease in threshold cycle value. Overall, our modified PCR method is simpler and more selective to the specific sites of damage in mtDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Genotoxicity of refinery waste assessed by some DNA damage tests.

    Science.gov (United States)

    Gupta, Amit Kumar; Ahmad, Irshad; Ahmad, Masood

    2015-04-01

    Refinery waste effluent is well known to contain polycyclic aromatic hydrocarbons, phenols and heavy metals as potentially genotoxic substances. The aim of the present study was to assess the genotoxic potential of Mathura refinery wastewater (MRWW) by various in vitro tests including the single cell gel electrophoresis, plasmid nicking assay and S1 nuclease assay. Treatment of human lymphocytes to different MRWW concentrations (0.15×, 0.3×, 0.5× and 0.78×) caused the formation of comets of which the mean tail lengths increased proportionately and differed significantly from those of unexposed controls. The toxic effect of MRWW on DNA was also studied by plasmid nicking assay and S1 nuclease assay. Strand breaks formation in the MRWW treated pBR322 plasmid confirmed its genotoxic effect. Moreover, a dose dependent increase in cleavage of calf thymus DNA in S1 nuclease assay was also suggestive of the DNA damaging potential of MRWW. A higher level of ROS generation in the test water sample was recorded which might be contributing to its genotoxicity. Interaction between the constituents of MRWW and calf thymus DNA was also ascertained by UV-visible spectroscopy.

  10. CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues.

    Science.gov (United States)

    Kava, Hieronimus W; Murray, Vincent

    2016-10-01

    This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use.

  11. Characterization of the target DNA sequence for the DNA-binding domain of zinc finger protein 191

    Institute of Scientific and Technical Information of China (English)

    Haoyue Wang; Ruilin Sun; Guoxiang Liu; Minghui Yao; Jian Fei; Hebai Shen

    2008-01-01

    Studies on the DNA-binding properties of transcription factors are important in searching for the downstream genes regulated by these factors. In the present study, we report on the DNA-binding property of a Cys2His2-type transcription factor, zinc finger protein 191 (Zfp191), which has been newly found to play a significant role in mice.By constructing a fusion protein containing the DNA-binding domain of Zfp191,we characterized target DNA by determining the protein's binding specificity and dependence on zinc.The data showed that the DNA-binding domain of Zfp191can specifically bind to the TCAT repeat motif and that there is a cooperative effect among the target DNA's multiple binding sites.Furthermore,the binding reaction is dependent on zinc.This work provides a foundation for further studies on the role of Zfp191 in gene regulation and development.

  12. Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein.

    Science.gov (United States)

    Sandetskaya, Natalia; Naumann, Andreas; Hennig, Katharina; Kuhlmeier, Dirk

    2014-06-01

    Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4 × 10(6)-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.

  13. Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

    Directory of Open Access Journals (Sweden)

    Yuki Nakayama

    Full Text Available The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA. Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR, which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1 DNA extracted from fresh frozen liver tissues (Frozen-DNA; 2 DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA; and 3 DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA. These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method

  14. Experimental strategies for studying transcription factor-DNA binding specificities.

    Science.gov (United States)

    Geertz, Marcel; Maerkl, Sebastian J

    2010-12-01

    Specific binding of transcription factors (TFs) determines in a large part the connectivity of gene regulatory networks as well as the quantitative level of gene expression. A multiplicity of both experimental and computational methods is currently used to discover and characterize the underlying TF-DNA interactions. Experimental methods can be further subdivided into in vitro- and in vivo-based approaches, each accenting different aspects of TF-binding events. In this review we summarize the flexibility and performance of a selection of both types of experimental methods. In conclusion, we argue that a serial combination of methods with different throughput and data type constitutes an optimal experimental strategy.

  15. Voltammetric Detection of Damage to DNA by Arsenic Compounds at a DNA Biosensor

    Directory of Open Access Journals (Sweden)

    R. Wennrich

    2005-11-01

    Full Text Available DNA biosensor can serve as a powerfull tool for simple in vitro tests of chemicaltoxicity. In this paper, damage to DNA attached to the surface of screen-printed carbonelectrode by arsenic compounds in solution is described. Using the Co(III complex with1,10-phenanthroline, [Co(phen3]3+ , as an electrochemical DNA marker and the Ru(IIcomplex with bipyridyne, [Ru(bipy3]2+ , as a DNA oxidation catalyst, the portion of originaldsDNA which survives an incubation of the biosensor in the cleavage medium was evaluated.The model cleavage mixture was composed of an arsenic compound at 10-3 mol/Lconcentration corresponding to real contaminated water, 2x10-4 mol/L Fe(II or Cu(II ions asthe redox catalyst, and 1.5x10-2 mol/L hydrogen peroxide. DNA damage by arsenite,dimethylarsinic acid as the metabolic product of inorganic arsenic and widely used herbicide,as well as phenylarsonic acid and p-arsanilic acid as the representatives of feed additives wasfound in difference to arsenate.

  16. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    Science.gov (United States)

    Shokri, Leila; Rouzina, Ioulia; Williams, Mark C.

    2009-06-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork.

  17. DBD2BS: connecting a DNA-binding protein with its binding sites.

    Science.gov (United States)

    Chien, Ting-Ying; Lin, Chih-Kang; Lin, Chih-Wei; Weng, Yi-Zhong; Chen, Chien-Yu; Chang, Darby Tien-Hao

    2012-07-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein-DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD-DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein-DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw.

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

    Science.gov (United States)

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

    2016-01-01

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

  19. Genomic DNA binding to ZnO microrods

    Science.gov (United States)

    Guzmán-Embús, D. A.; Cardozo, M. Orrego; Vargas-Hernández, C.

    2015-08-01

    In this work, ZnO microrods were produced by hydrothermal synthesis. DNA was extracted from pork spleen cells by cellular lysis, deproteinization and precipitation. The analysis of the DNA binding to the ZnO was performed using Raman spectroscopy a technique that allowed for the evaluation of the effect that the presence of the ZnO in the complex has on the DNA structure. Vibrational spectral bands from the DNA molecule and hexagonal wurtzite ZnO were observed and classified as E2(M), A1(TO), E2(High), E1(LO) and 2LO. The Raman signals from the vibrational bands corresponding to the phosphodiester bond 5‧-C-O-P-O-C-3‧ and bond stretching of the PO2- group, as well as ring vibrations of the nitrogenous bases of the DNA, were enhanced by the presence of the ZnO microrods. The bands from the modes corresponding to the C-O and Odbnd Psbnd O- molecules of the DNA backbone were observed to exhibit larger spectral shifts due to the compression and tensile stresses generated at the ZnO/DNA interface, respectively. In addition, the relative vibrational mode intensities of the nitrogenous bases increased.

  20. Elasticity of DNA and the effect of Dendrimer Binding

    CERN Document Server

    Mogurampelly, Santosh; Netz, Roland R; Maiti, Prabal K

    2013-01-01

    Negatively charged DNA can be compacted by positively charged dendrimers and the degree of compaction is a delicate balance between the strength of the electrostatic interaction and the elasticity of DNA. We report various elastic properties of short double stranded DNA (dsDNA) and the effect of dendrimer binding using fully atomistic molecular dynamics and numerical simulations. In equilibrium at room temperature, the contour length distribution P(L) and end-to-end distance distribution P(R) are nearly Gaussian, the former gives an estimate of the stretch modulus {\\gamma}_1 of dsDNA in quantitative agreement with the literature value. The bend angle distribution P({\\theta}) of the dsDNA also has a Gaussian form and allows to extract a persistence length, L_p of 43 nm. When the dsDNA is compacted by positively charged dendrimer, the stretch modulus stays invariant but the effective bending rigidity estimated from the end-to-end distance distribution decreases dramatically due to backbone charge neutralization...

  1. DNA Damage by Radiation in Tradescantia Leaf Cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-15

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

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

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

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

  3. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    Science.gov (United States)

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J

    2011-10-01

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

  4. Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

    Science.gov (United States)

    Wooddell, Christine I; Subbotin, Vladimir M; Sebestyén, Magdolna G; Griffin, Jacob B; Zhang, Guofeng; Schleef, Martin; Braun, Serge; Huss, Thierry; Wolff, Jon A

    2011-02-01

    Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

  5. Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP.

    Science.gov (United States)

    Chao, C C; Sun, N K; Lin-Chao, S

    1993-02-15

    A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

  6. DDB2 association with PCNA is required for its degradation after UV-induced DNA damage.

    Science.gov (United States)

    Cazzalini, Ornella; Perucca, Paola; Mocchi, Roberto; Sommatis, Sabrina; Prosperi, Ennio; Stivala, Lucia Anna

    2014-01-01

    DDB2 is a protein playing an essential role in the lesion recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER) process. Among the proteins involved in the DNA damage response, p21(CDKN1A) (p21) has been reported to participate in NER, but also to be removed by proteolytic degradation, thanks to its association with PCNA. DDB2 is involved in the CUL4-DDB1 complex mediating p21 degradation; however, the direct interaction between DDB2, p21 and PCNA has been never investigated. Here, we show that DDB2 co-localizes with PCNA and p21 at local UV-induced DNA-damage sites, and these proteins co-immunoprecipitate in the same complex. In addition, we provide evidence that p21 is not able to bind directly DDB2, but, to this end, the presence of PCNA is required. Direct physical association of recombinant DDB2 protein with PCNA is mediated by a conserved PIP-box present in the N-terminal region of DDB2. Mutation of the PIP-box resulted in the loss of protein interaction. Interestingly, the same mutation, or depletion of PCNA by RNA interference, greatly impaired DDB2 degradation induced by UV irradiation. These results indicate that DDB2 is a PCNA-binding protein, and that this association is required for DDB2 proteolytic degradation.

  7. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

    Science.gov (United States)

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

    2012-09-15

    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Potentially lethal damage repair by total and quiescent tumor cells following various DNA-damaging treatments

    Energy Technology Data Exchange (ETDEWEB)

    Masunaga, Shin-ichiro; Ono, Koji; Suzuki, Minoru; Kinashi, Yuko; Takagaki, Masao [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst; Hori, Hitoshi; Kasai, Soko; Nagasawa, Hideko; Uto, Yoshihiro

    1999-08-01

    After continuous labeling of proliferating (P) cells with 5-bromo-2'-deoxyuridine (BrdU) for 5 days, SCC VII tumor-bearing mice received various kinds of DNA-damaging treatments: gamma-ray irradiation, tirapazamine (TPZ, hypoxia-specific cytotoxin) administration, or cisplatin injection. From 0.5 to 72 hr after treatment, tumors were excised, minced, and trypsinized. Single tumor cell suspensions were incubated for 48 hr with a cytokinesis-blocker, cytochalasin-B. Then, the micronucleus (MN) frequency for BrdU-unlabeled cells, quiescent (Q) cells at treatment, was determined using immunofluorescence staining for BrdU. The MN frequency for total (P+Q) cells was obtained from tumors that were not pretreated with BrdU labeling. The sensitivity to each DNA-damaging treatment was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). Treatment with gamma-rays or cisplatin resulted in a larger MN frequency in total cells than in Q cells. In contrast, TPZ treatment produced a smaller MN frequency in total cells than in Q cells. Regardless of the treatment used, Q cells showed greater repair capacities than total cells. However, TPZ caused much smaller repair capacity in both total and Q cells, compared with gamma-rays or cisplatin. Gamma-rays and cisplatin produced similar repair patterns. Differences in sensitivity between total and Q cells and repair patterns of the two cell populations were thought to depend on differences between the two cell populations in the toxicity of the DNA-damaging treatment and distribution pattern of the anticancer agent. (author)

  9. Synthesis, DNA binding and topoisomerase inhibition of mononaphthalimide homospermidine derivatives

    Institute of Scientific and Technical Information of China (English)

    Zhi Yong Tian; Hong Xia Ma; Song Qiang Xie; Xue Wang; Jin Zhao; Chao Jie Wang; Wen Yuan Gao

    2008-01-01

    Two novel mononaphthalimide homospermidine derivatives (2a, 2b) with three or four methylene unit as linkages weresynthesized and evaluated for cytotoxicity against human leukemia K562, murine melanoma B 16 and Chinese hamster ovary CHOcell lines. The presence of homospermidine motif could greatly elevate the potency of 1,8-naphthalimide. Conjugate 2b with longerspacer exhibited higher in vitro cytotoxicity than 2a. The DNA binding experiments indicated that conjugates 2b could bind toherring sperm DNA. The topoisomerase Ⅱ poison trials revealed that 2b could inhibit the activity of top. Ⅱ.2008 Chao Jie Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  10. A Single-Molecule Study on the Structural Damage of Ultraviolet Radiated DNA

    Directory of Open Access Journals (Sweden)

    Pu Chun Ke

    2008-04-01

    Full Text Available The structural damage of double-stranded DNA under UV radiation was examined using single-molecule fluorescence microscopy. Compared to undamaged DNA, the diffusion coefficient of λ-DNA was significantly increased with 12 min or 20 min of radiation but remained unchanged for 40 min of exposure possibly due to strand crosslinking. The structural damage of DNA was further examined using transmission electron microscopy which revealed kinks and sharp bends along the DNA backbone.

  11. Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation.

    Directory of Open Access Journals (Sweden)

    Xurui Zhang

    Full Text Available Persistent DNA damage is considered as a main cause of cellular senescence induced by ionizing radiation. However, the molecular bases of the DNA damage and their contribution to cellular senescence are not completely clear. In this study, we found that both heavy ions and X-rays induced senescence in human uveal melanoma 92-1 cells. By measuring senescence associated-β-galactosidase and cell proliferation, we identified that heavy ions were more effective at inducing senescence than X-rays. We observed less efficient repair when DNA damage was induced by heavy ions compared with X-rays and most of the irreparable damage was complex of single strand breaks and double strand breaks, while DNA damage induced by X-rays was mostly repaired in 24 hours and the remained damage was preferentially associated with telomeric DNA. Our results suggest that DNA damage induced by heavy ion is often complex and difficult to repair, thus presents as persistent DNA damage and pushes the cell into senescence. In contrast, persistent DNA damage induced by X-rays is preferentially associated with telomeric DNA and the telomere-favored persistent DNA damage contributes to X-rays induced cellular senescence. These findings provide new insight into the understanding of high relative biological effectiveness of heavy ions relevant to cancer therapy and space radiation research.

  12. Autoantigenic proteins that bind recombinogenic sequences in Epstein-Barr virus and cellular DNA.

    OpenAIRE

    1994-01-01

    We have identified conserved autoantigenic cellular proteins that bind to G-rich sequence motifs in recombinogenic regions of Epstein-Barr virus (EBV) DNA. This binding activity, called TRBP, recognizes the EBV terminal repeats, a locus responsible for interconversion of linear and circular EBV DNA. We found that TRBP also binds to EBV DNA sequences involved in deletion of EBNA2, a gene product required for immortalization. We show that TRBP binds sequences present in repetitive cellular DNA,...

  13. Interplay between DNA tumor viruses and the host DNA damage response.

    Science.gov (United States)

    McFadden, Karyn; Luftig, Micah A

    2013-01-01

    Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.

  14. ELF alternating magnetic field decreases reproduction by DNA damage induction.

    Science.gov (United States)

    Panagopoulos, Dimitris J; Karabarbounis, Andreas; Lioliousis, Constantinos

    2013-11-01

    In the present experiments, the effect of 50-Hz alternating magnetic field on Drosophila melanogaster reproduction was studied. Newly eclosed insects were separated into identical groups of ten males and ten females and exposed to three different intensities of the ELF magnetic field (1, 11, and 21 G) continuously during the first 5 days of their adult lives. The reproductive capacity was assessed by the number of F1 pupae according to a well-defined protocol of ours. The magnetic field was found to decrease reproduction by up to 4.3%. The effect increased with increasing field intensities. The decline in reproductive capacity was found to be due to severe DNA damage (DNA fragmentation) and consequent cell death induction in the reproductive cells as determined by the TUNEL assay applied during early and mid-oogenesis (from germarium to stage 10) where physiological apoptosis does not occur. The increase in DNA damage was more significant than the corresponding decrease in reproductive capacity (up to ~7.5%). The TUNEL-positive signal denoting DNA fragmentation was observed exclusively at the two most sensitive developmental stages of oogenesis: the early and mid-oogenesis checkpoints (i.e. region 2a/2b of the germarium and stages 7-8 just before the onset of vitellogenesis)-in contrast to exposure to microwave radiation of earlier work of ours in which the DNA fragmentation was induced at all developmental stages of early and mid-oogenesis. Moreover, the TUNEL-positive signal was observed in all three types of egg chamber cells, mainly in the nurse and follicle cells and also in the oocyte, in agreement with the microwave exposure of our earlier works. According to previous reports, cell death induction in the oocyte was observed only in the case of microwave exposure and not after exposure to other stress factors as toxic chemicals or food deprivation. Now it is also observed for the first time after ELF magnetic field exposure. Finally, in contrast to microwave

  15. Effect of DNA binding protein Ssh12 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

    Institute of Scientific and Technical Information of China (English)

    楼慧强; 黄力; VietQ.Mai

    1999-01-01

    An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.

  16. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1.

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

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

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

  18. Host DNA damage response facilitates African swine fever virus infection.

    Science.gov (United States)

    Simões, Margarida; Martins, Carlos; Ferreira, Fernando

    2013-07-26

    Studies with different viral infection models on virus interactions with the host cell nucleus have opened new perspectives on our understanding of the molecular basis of these interactions in African swine fever virus (ASFV) infection. The present study aims to characterize the host DNA damage response (DDR) occurring upon in vitro infection with the ASFV-Ba71V isolate. We evaluated protein levels during ASFV time-course infection, of several signalling cascade factors belonging to DDR pathways involved in double strand break repair - Ataxia Telangiectasia Mutated (ATM), ATM-Rad 3 related (ATR) and DNA dependent protein kinase catalytic subunit (DNA-PKcs). DDR inhibitory trials using caffeine and wortmannin and ATR inducible-expression cell lines were used to confirm specific pathway activation during viral infection. Our results show that ASFV specifically elicits ATR-mediated pathway activation from the early phase of infection with increased levels of H2AX, RPA32, p53, ATR and Chk1 phosphorylated forms. Viral p72 synthesis was abrogated by ATR kinase inhibitors and also in ATR-kd cells. Furthermore, a reduction of viral progeny was identified in these cells when compared to the outcome of infection in ATR-wt. Overall, our results strongly suggest that the ATR pathway plays an essential role for successful ASFV infection of host cells.

  19. Dynamics of the human nuclear proteome in response to DNA damage

    NARCIS (Netherlands)

    Dirksen, Eef Hubert Cecil

    2006-01-01

    The genome is constantly challenged by factors that can induce DNA damage and thereby threaten the viability of the cell. If DNA damage remains unrepaired it can lead to the development of cancer. Although much is known about the role of proteins and protein complexes in the cellular response to DNA

  20. 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 left unre

  1. Mitochondrial DNA damage: Molecular marker of vulnerable nigral neurons in Parkinson's disease

    NARCIS (Netherlands)

    L.H. Sanders (Laurie); J. McCoy (Jennifer); X. Hu (Xiaoping); P.G. Mastroberardino (Pier); B.C. Dickinson (Bryan); C.J. Chang (Christopher); C.T. Chu (Charleen); B. van Houten (Bennett); J.T. Greenamyre (Timothy)

    2014-01-01

    textabstractDNA damage can cause (and result from) oxidative stress and mitochondrial impairment, both of which are implicated in the pathogenesis of Parkinson's disease (PD). We therefore examined the role of mitochondrial DNA (mtDNA) damage in human postmortem brain tissue and in in vivo and in vi

  2. 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

  3. Mitochondrial DNA damage: Molecular marker of vulnerable nigral neurons in Parkinson's disease

    NARCIS (Netherlands)

    L.H. Sanders (Laurie); J. McCoy (Jennifer); X. Hu (Xiaoping); P.G. Mastroberardino (Pier); B.C. Dickinson (Bryan); C.J. Chang (Christopher); C.T. Chu (Charleen); B. van Houten (Bennett); J.T. Greenamyre (Timothy)

    2014-01-01

    textabstractDNA damage can cause (and result from) oxidative stress and mitochondrial impairment, both of which are implicated in the pathogenesis of Parkinson's disease (PD). We therefore examined the role of mitochondrial DNA (mtDNA) damage in human postmortem brain tissue and in in vivo and in

  4. Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

    Science.gov (United States)

    Necchi, Daniela; Pinto, Antonella; Tillhon, Micol; Dutto, Ilaria; Serafini, Melania Maria; Lanni, Cristina; Govoni, Stefano; Racchi, Marco; Prosperi, Ennio

    2015-10-01

    Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase β, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors.

  5. Computational redesign of endonuclease DNA binding and cleavage specificity

    Science.gov (United States)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  6. TRF2 binds branched DNA to safeguard telomere integrity.

    Science.gov (United States)

    Schmutz, Isabelle; Timashev, Leonid; Xie, Wei; Patel, Dinshaw J; de Lange, Titia

    2017-09-01

    Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

  7. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    Science.gov (United States)

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  8. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ruthenium-fluoroquinolone complexes

    Indian Academy of Sciences (India)

    Mohan N Patel; Hardik N Joshi; Chintan R Patel

    2014-05-01

    Six new Ru(II) and Ru(III) complexes have been synthesized and characterized by elemental analysis, LC-MS, electronic spectra, IR spectra and magnetic moment measurements. DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as their HS DNA cleavage properties by means of agarose gel electrophoresis. The experimental results show that all the complexes can bind to DNA via partial intercalative mode. The b values of complexes were found in the range 2.14 × 104 to 2.70 × 105 M-1. All the complexes show excellent efficiency of cleaving DNA than respective fluoroquinolones. Brine shrimp lethality bioassay has been performed to check the cytotoxic activity. The IC50 values of the complexes are in the range of 6.27 to 16.05 g mL-1.

  9. Phosphorylation-dependent interactions between Crb2 and Chk1 are essential for DNA damage checkpoint.

    Directory of Open Access Journals (Sweden)

    Meng Qu

    2012-07-01

    Full Text Available In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1 complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.

  10. A novel DNA-binding domain in the Shrunken initiator-binding protein (IBP1).

    Science.gov (United States)

    Lugert, T; Werr, W

    1994-06-01

    South-western screening of lambda gt11 expression library with a fragment of the Shrunken promoter containing the initiator element resulted in cloning of a novel maize gene. The encoded initiator-binding protein (IBP1) interacts at the transcription start site of the Shrunken promoter. Analysis of the 680 amino acid (aa) long polypeptide revealed a novel bipartite DNA-binding domain at the carboxyl terminus. In its amino-terminal part, it is weakly related to Myb R-repeats but the following basic region is also essential for DNA binding. A region of similarity to the conserved 2.1 and 2.2 motifs in bacterial sigma-factors is located close to the IBP1 amino terminus. Two putative nuclear localization signals are compatible with the presence of antigenically related polypeptides in nuclear protein extracts. The IBP1 gene was mapped to the long arm of chromosome 9 (9L095); a second highly related gene IBP2 is located on the short arm of chromosome 1 (1S014). Both genes encode proteins sharing 93% similarity and are transcribed with similar activity in different plant organs. A small 82 nucleotide intron in the IBP2 transcript is found unspliced to a variable degree in different tissues. Translation of this incompletely processed transcript would result in a truncated amino-terminal polypeptide lacking the DNA-binding domain.

  11. Mdb1, a fission yeast homolog of human MDC1, modulates DNA damage response and mitotic spindle function.

    Directory of Open Access Journals (Sweden)

    Yi Wei

    Full Text Available During eukaryotic DNA damage response (DDR, one of the earliest events is the phosphorylation of the C-terminal SQ motif of histone H2AX (H2A in yeasts. In human cells, phosphorylated H2AX (γH2AX is recognized by MDC1, which serves as a binding platform for the accumulation of a myriad of DDR factors on chromatin regions surrounding DNA lesions. Despite its important role in DDR, no homolog of MDC1 outside of metazoans has been described. Here, we report the characterization of Mdb1, a protein from the fission yeast Schizosaccharomyces pombe, which shares significant sequence homology with human MDC1 in their C-terminal tandem BRCT (tBRCT domains. We show that in vitro, recombinant Mdb1 protein binds a phosphorylated H2A (γH2A peptide, and the phospho-specific binding requires two conserved phospho-binding residues in the tBRCT domain of Mdb1. In vivo, Mdb1 forms nuclear foci at DNA double strand breaks (DSBs induced by the HO endonuclease and ionizing radiation (IR. IR-induced Mdb1 focus formation depends on γH2A and the phospho-binding residues of Mdb1. Deleting the mdb1 gene does not overtly affect DNA damage sensitivity in a wild type background, but alters the DNA damage sensitivity of cells lacking another γH2A binder Crb2. Overexpression of Mdb1 causes severe DNA damage sensitivity in a manner that requires the interaction between Mdb1 and γH2A. During mitosis, Mdb1 localizes to spindles and concentrates at spindle midzones at late mitosis. The spindle midzone localization of Mdb1 requires its phospho-binding residues, but is independent of γH2A. Loss of Mdb1 or mutating its phospho-binding residues makes cells more resistant to the microtubule depolymerizing drug thiabendazole. We propose that Mdb1 performs dual roles in DDR and mitotic spindle regulation.

  12. Association of DNA damage and dyslipidemia with polycystic ovarian syndrome

    Directory of Open Access Journals (Sweden)

    Manikkumar R

    2013-02-01

    Full Text Available Polycystic ovary syndrome (PCOS is associated with hyperinsuli-nemia and insulin resistance which may lead to cardiovascular diseases. Evidence for cardiovascular events in women who were affected by PCOS during fertile age is limited. The pathogenesis is unknown; however, it is a complex multigenetic disorder. The present study was undertaken to evaluate the various cardiovas-cular risk factors and their DNA repair efficiency in women with PCOS by investigating the biochemical, endocrinological and mo-lecular cytogenetic alterations. These investigations were carried out in 116 women in the age group of 15-35 years clinically diag-nosed with PCOS. Data were compared with that of 50 age-matched healthy normal women. Fasting blood sugar (FBS, Lipid profile, Follicle-Stimulating Hormone (FSH and Luteinizing Hor-mone (LH, Prolactin and Estradiol were estimated after getting the informed consent. Mutagen induced chromosome sensitivity analysis was carried out in the lymphocytes of the subjects to as-sess the DNA repair proficiency. Fasting Blood Sugar, total cho-lesterol and LDL cholesterol were found to be elevated whereas HDL cholesterol was found to be lowered in the test subjects. FSH, LH and prolactin were also found to be significantly elevated in the test subjects. Change in the estradiol concentration in the test subjects was not significant. The mutagen sensitivity analysis revealed a significant elevation in break per cell (b/c values indi-cating a deficiency in the DNA repair mechanism / DNA damage in PCOS patients. Modification of life style by changing the dietary habit and sedentary life style will help to reduce the oxidative stress and may increase the ovarian function and a sensible life-style management is recommended for reducing the risk for CVD.

  13. Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA.

    Directory of Open Access Journals (Sweden)

    John F Domsic

    Full Text Available LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

  14. Visualizing the search for radiation-damaged DNA bases in real time

    Science.gov (United States)

    Lee, Andrea J.; Wallace, Susan S.

    2016-11-01

    The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

  15. p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo

    Directory of Open Access Journals (Sweden)

    Oleg Timofeev

    2013-05-01

    Full Text Available Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

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

    Science.gov (United States)

    Taylor, John-Stephen

    2015-12-01

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

  17. Oxidative DNA damage after transplantation of the liver and small intestine in pigs

    DEFF Research Database (Denmark)

    Loft, S; Larsen, P N; Rasmussen, A

    1995-01-01

    Oxidative damage is thought to play an important role in ischemia/reperfusion injury, including the outcome of transplantation of the liver and intestine. We have investigated oxidative DNA damage after combined transplantation of the liver and small intestine in 5 pigs. DNA damage was estimated...... to DNA results from reperfusion of transplanted small intestine and liver in pigs, as estimated from the readily excreted repair product 8-oxodG....

  18. Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2)

    OpenAIRE

    Pérez-Caro, M.; Bermejo-Rodríguez, C.; González-Herrero, I; Sánchez-Beato, M; Piris, M. A.; Sánchez-García, I

    2008-01-01

    Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in respon...

  19. Regulation of DNA Damage Response by Estrogen Receptor β-Mediated Inhibition of Breast Cancer Associated Gene 2

    Directory of Open Access Journals (Sweden)

    Yuan-Hao Lee

    2015-04-01

    Full Text Available Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2, correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

  20. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Science.gov (United States)

    Schrader, Anna; Gross, Thomas; Thalhammer, Verena; Längst, Gernot

    2015-01-01

    The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  1. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Directory of Open Access Journals (Sweden)

    Anna Schrader

    Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  2. Direct detection and quantification of abasic sites for in vivo studies of DNA damage and repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang Yanming [Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University, Cleveland, OH 44122 (United States)], E-mail: yanming.wang@case.edu; Liu Lili [Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122 (United States); Wu Chunying [Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University, Cleveland, OH 44122 (United States); Bulgar, Alina [Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122 (United States); Somoza, Eduardo; Zhu Wenxia [Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University, Cleveland, OH 44122 (United States); Gerson, Stanton L. [Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122 (United States)

    2009-11-15

    Use of chemotherapeutic agents to induce cytotoxic DNA damage and programmed cell death is a key strategy in cancer treatments. However, the efficacy of DNA-targeted agents such as temozolomide is often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway resulted in formation of abasic or apurinic/apyrimidinic (AP) sites, and blockage of AP sites led to a significant enhancement of drug sensitivity due to reduction of DNA base excision repair. Since a number of chemotherapeutic agents also induce formation of AP sites, monitoring of these sites as a clinical correlate of drug effect will provide a useful tool in the development of DNA-targeted chemotherapies aimed at blocking abasic sites from repair. Here we report an imaging technique based on positron emission tomography (PET) that allows for direct quantification of AP sites in vivo. For this purpose, positron-emitting carbon-11 has been incorporated into methoxyamine ([{sup 11}C]MX) that binds covalently to AP sites with high specificity. The binding specificity of [{sup 11}C]MX for AP sites was demonstrated by in vivo blocking experiments. Using [{sup 11}C]MX as a radiotracer, animal PET studies have been conducted in melanoma and glioma xenografts for quantification of AP sites. Following induction of AP sites by temozolomide, both tumor models showed significant increase of [{sup 11}C]MX uptake in tumor regions in terms of radioactivity concentration as a function of time, which correlates well with conventional aldehyde reactive probe (ARP)-based bioassays for AP sites.

  3. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Science.gov (United States)

    Kitange, Gaspar J.; Mladek, Ann C.; Schroeder, Mark A.; Pokorny, Jenny C.; Carlson, Brett L.; Zhang, Yuji; Nair, Asha A.; Lee, Jeong-Heon; Yan, Huihuang; Decker, Paul A.; Zhang, Zhiguo; Sarkaria, Jann N.

    2016-01-01

    Summary Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of 2 key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51 and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin modifying complex that binds within the promoter of MGMT, RAD51 and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. RBBP4/CBP/p300 complex may provide an interesting target for developing therapy sensitizing strategies for GBM and other tumors. PMID:26972001

  4. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Directory of Open Access Journals (Sweden)

    Gaspar J. Kitange

    2016-03-01

    Full Text Available Here we provide evidence that RBBP4 modulates temozolomide (TMZ sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac and increased H3K9 tri-methylation (H3K9me3. Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.

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

    Science.gov (United States)

    Ganesan, Shanthi; Keating, Aileen F

    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 6μM) for 24 or 48h. Cell viability was reduced (Padduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G., E-mail: lhudson@salud.unm.edu

    2013-06-01

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

  7. Poetry in motion: Increased chromosomal mobility after DNA damage.

    Science.gov (United States)

    Smith, Michael J; Rothstein, Rodney

    2017-08-01

    Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy.

    Science.gov (United States)

    Fayzullina, Saniya; Martin, Lee J

    2016-09-01

    We studied DNA damage response (DDR) and DNA repair capacities of skeletal muscle cells from a mouse model of infantile spinal muscular atrophy (SMA) caused by loss-of-function mutation of survival of motor neuron (Smn). Primary myocyte cultures derived from skeletal muscle satellite cells of neonatal control and mutant SMN mice had similar myotube length, myonuclei, satellite cell marker Pax7 and differentiated myotube marker myosin, and acetylcholine receptor clustering. DNA damage was induced in differentiated skeletal myotubes by γ-irradiation, etoposide, and methyl methanesulfonate (MMS). Unexposed control and SMA myotubes had stable genome integrity. After γ-irradiation and etoposide, myotubes repaired most DNA damage equally. Control and mutant myotubes exposed to MMS exhibited equivalent DNA damage without repair. Control and SMA myotube nuclei contained DDR proteins phospho-p53 and phospho-H2AX foci that, with DNA damage, dispersed and then re-formed similarly after recovery. We conclude that mouse primary satellite cell-derived myotubes effectively respond to and repair DNA strand-breaks, while DNA alkylation repair is underrepresented. Morphological differentiation, genome stability, genome sensor, and DNA strand-break repair potential are preserved in mouse SMA myocytes; thus, reduced SMN does not interfere with myocyte differentiation, genome integrity, and DNA repair, and faulty DNA repair is unlikely pathogenic in SMA.

  9. Critical functions of Rpa3/Ssb3 in S-phase DNA damage responses in fission yeast.

    Directory of Open Access Journals (Sweden)

    Santiago Cavero

    2010-09-01

    Full Text Available Replication Protein A (RPA is a heterotrimeric, single-stranded DNA (ssDNA-binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11Rpa1 and Rad22Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.

  10. R-Roscovitine (Seliciclib prevents DNA damage-induced cyclin A1 upregulation and hinders non-homologous end-joining (NHEJ DNA repair

    Directory of Open Access Journals (Sweden)

    Federico Mario

    2010-08-01

    Full Text Available Abstract Background CDK-inhibitors can diminish transcriptional levels of cell cycle-related cyclins through the inhibition of E2F family members and CDK7 and 9. Cyclin A1, an E2F-independent cyclin, is strongly upregulated under genotoxic conditions and functionally was shown to increase NHEJ activity. Cyclin A1 outcompetes with cyclin A2 for CDK2 binding, possibly redirecting its activity towards DNA repair. To see if we could therapeutically block this switch, we analyzed the effects of the CDK-inhibitor R-Roscovitine on the expression levels of cyclin A1 under genotoxic stress and observed subsequent DNA damage and repair mechanisms. Results We found that R-Roscovitine alone was unable to alter cyclin A1 transcriptional levels, however it was able to reduce protein expression through a proteosome-dependent mechanism. When combined with DNA damaging agents, R-Roscovitine was able to prevent the DNA damage-induced upregulation of cyclin A1 on a transcriptional and post-transcriptional level. This, moreover resulted in a significant decrease in non-homologous end-joining (NHEJ paired with an increase in DNA DSBs and overall DNA damage over time. Furthermore, microarray analysis demonstrated that R-Roscovitine affected DNA repair mechanisms in a more global fashion. Conclusions Our data reveal a new mechanism of action for R-Roscovitine on DNA repair through the inhibition of the molecular switch between cyclin A family members under genotoxic conditions resulting in reduced NHEJ capability.

  11. Phase Behavior of DNA in the Presence of DNA-Binding Proteins.

    Science.gov (United States)

    Le Treut, Guillaume; Képès, François; Orland, Henri

    2016-01-01

    To characterize the thermodynamical equilibrium of DNA chains interacting with a solution of nonspecific binding proteins, we implemented a Flory-Huggins free energy model. We explored the dependence on DNA and protein concentrations of the DNA collapse. For physiologically relevant values of the DNA-protein affinity, this collapse gives rise to a biphasic regime with a dense and a dilute phase; the corresponding phase diagram was computed. Using an approach based on Hamiltonian paths, we show that the dense phase has either a molten globule or a crystalline structure, depending on the DNA bending rigidity, which is influenced by the ionic strength. These results are valid at the thermodynamical equilibrium and therefore should be consistent with many biological processes, whose characteristic timescales range typically from 1 ms to 10 s. Our model may thus be applied to biological phenomena that involve DNA-binding proteins, such as DNA condensation with crystalline order, which occurs in some bacteria to protect their chromosome from detrimental factors; or transcription initiation, which occurs in clusters called transcription factories that are reminiscent of the dense phase characterized in this study.

  12. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B [Z; (W Elec.); (NCSU)

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

  13. 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.

  14. A robust assay to measure DNA topology-dependent protein binding affinity.

    Science.gov (United States)

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.

  15. Effect of low energy electron irradiation on DNA damage by Cu{sup 2+} ion

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Hyung Ah; Cho, Hyuck [Dept. of Physics, Chungnam National University, Daejeon (Korea, Republic of); Park, Yeun Soo [Plasma Technology Research Center, National Fusion Research Institute, Gunsan (Korea, Republic of)

    2017-03-15

    The combined effect of the low energy electron (LEE) irradiation and Cu{sup 2+} ion on DNA damage was investigated. Lyophilized pBR322 plasmid DNA films with various concentrations (1–15 mM) of Cu{sup 2+} ion were independently irradiated by monochromatic LEEs with 5 eV. The types of DNA damage, single strand break (SSB) and double strand break (DSB), were separated and quantified by gel electrophoresis. Without electron irradiation, DNA damage was slightly increased with increasing Cu ion concentration via Fenton reaction. LEE-induced DNA damage, with no Cu ion, was only 6.6% via dissociative electron attachment (DEA) process. However, DNA damage was significantly increased through the combined effect of LEE-irradiation and Cu ion, except around 9 mM Cu ion. The possible pathways of DNA damage for each of these different cases were suggested. The combined effect of LEE-irradiation and Cu ion is likely to cause increasing dissociation after elevated transient negative ion state, resulting in the enhanced DNA damage. For the decrease of DNA damage at around 9-mM Cu ion, it is assumed to be related to the structural stabilization due to DNA inter- and intra-crosslinks via Cu ion.

  16. DNA Damage Response in Hematopoietic Stem Cell Ageing.

    Science.gov (United States)

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

    2016-06-01

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

  17. DNA Damage Response in Hematopoietic Stem Cell Ageing

    Directory of Open Access Journals (Sweden)

    Tangliang Li

    2016-06-01

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

  18. The DNA damage response in viral-induced cellular transformation.

    Science.gov (United States)

    Nikitin, P A; Luftig, M A

    2012-01-31

    The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

  19. DNA Damage Response in Hematopoietic Stem Cell Ageing

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

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

  20. Retinoblastoma loss modulates DNA damage response favoring tumor progression.

    Directory of Open Access Journals (Sweden)

    Marcos Seoane

    Full Text Available Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS, crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12. Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

  1. Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect

    Energy Technology Data Exchange (ETDEWEB)

    Zimmerman, S.B.; Harrison, B.

    1987-05-01

    Macromolecular crowding extends the range of ionic conditions supporting high DNA polymerase reaction rates. Reactions tested were nick translation and gap-filling by DNA polymerase I of E. coli, nuclease and polymerase activities of the large fragment of that polymerase, and polymerization by the T4 DNA polymerase. For all of these reactions, high concentrations of nonspecific polymers increased enzymatic activity under otherwise inhibitory conditions resulting from relatively high ionic strength. The primary mechanism of the polymer effect seems to be to increase the binding of polymerase to DNA. They suggest that this effect of protein-DNA complexes is only one example of a general metabolic buffering action of crowded solutions on a variety of macromolecular interactions.

  2. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    Science.gov (United States)

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.

  3. Prediction of Protein-DNA binding by Monte Carlo method

    Science.gov (United States)

    Deng, Yuefan; Eisenberg, Moises; Korobka, Alex

    1997-08-01

    We present an analysis and prediction of protein-DNA binding specificity based on the hydrogen bonding between DNA, protein, and auxillary clusters of water molecules. Zif268, glucocorticoid receptor, λ-repressor mutant, HIN-recombinase, and tramtrack protein-DNA complexes are studied. Hydrogen bonds are approximated by the Lennard-Jones potential with a cutoff distance between the hydrogen and the acceptor atoms set to 3.2 Åand an angular component based on a dipole-dipole interaction. We use a three-stage docking algorithm: geometric hashing that matches pairs of hydrogen bonding sites; (2) least-squares minimization of pairwise distances to filter out insignificant matches; and (3) Monte Carlo stochastic search to minimize the energy of the system. More information can be obtained from our first paper on this subject [Y.Deng et all, J.Computational Chemistry (1995)]. Results show that the biologically correct base pair is selected preferentially when there are two or more strong hydrogen bonds (with LJ potential lower than -0.20) that bind it to the protein. Predicted sequences are less stable in the case of weaker bonding sites. In general the inclusion of water bridges does increase the number of base pairs for which correct specificity is predicted.

  4. APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, Bret D.; Berman, Zachary; Mueller, Geoffrey A.; Lin, Yunfeng; Chang, Timothy; Andres, Sara N.; Wojtaszek, Jessica L.; DeRose, Eugene F.; Appel, C. Denise; London, Robert E.; Yan, Shan; Williams, R. Scott

    2016-12-27

    The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.

  5. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Srinivas, L.; Shalini, V.K. (Department of Nutrition and Food Safety, Central Food Technological Research Institute, Mysore (India))

    1991-01-01

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

  7. The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-specific DNA binding.

    Science.gov (United States)

    Streaker, Emily D; Gupta, Aditi; Beckett, Dorothy

    2002-12-03

    The Escherichia coli biotin repressor, an allosteric transcriptional regulator, is activated for binding to the biotin operator by the small molecule biotinyl-5'-AMP. Results of combined thermodynamic, kinetic, and structural studies of the protein have revealed that corepressor binding results in disorder to order transitions in the protein monomer that facilitate tighter dimerization. The enhanced stability of the dimer leads to stabilization of the resulting biotin repressor-biotin operator complex. It is not clear, however, that the allosteric response in the system is transmitted solely through the protein-protein interface. In this work, the allosteric mechanism has been quantitatively probed by measuring the biotin operator binding and dimerization properties of three biotin repressor species: the apo or unliganded form, the biotin-bound form, and the holo or bio-5'-AMP-bound form. Comparisons of the pairwise differences in the bioO binding and dimerization energetics for the apo and holo species reveal that the enhanced DNA binding energetics resulting from adenylate binding track closely with the enhanced assembly energetics. However, when the results for repressor pairs that include the biotin-bound species are compared, no such equivalence is observed.

  8. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    Science.gov (United States)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  9. Curcusone C induces telomeric DNA-damage response in cancer cells through inhibition of telomeric repeat factor 2.

    Science.gov (United States)

    Wang, Mingxue; Cao, Jiaojiao; Zhu, Jian-Yong; Qiu, Jun; Zhang, Yan; Shu, Bing; Ou, Tian-Miao; Tan, Jia-Heng; Gu, Lian-Quan; Huang, Zhi-Shu; Yin, Sheng; Li, Ding

    2017-11-01

    Telomeric repeat factor 2 (known as TRF2 or TERF2) is a key component of telomere protection protein complex named as Shelterin. TRF2 helps the folding of telomere to form T-loop structure and the suppression of ATM-dependent DNA damage response activation. TRF2 has been recognized as a potentially new therapeutic target for cancer treatment. In our routine screening of small molecule libraries, we found that Curcusone C had significant effect in disrupting the binding between TRF2 and telomeric DNA, with potent antitumor activity against cancer cells. Our result showed that Curcusone C could bind with TRF2 without binding interaction with TRF1 (telomeric repeat factor 1) although these two