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Sample records for dna base excision

  1. Envisioning the molecular choreography of DNA base excision repair.

    PubMed

    Parikh, S S; Mol, C D; Hosfield, D J; Tainer, J A

    1999-02-01

    Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.

  2. Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

    PubMed Central

    Zhu, Chenxu; Lu, Lining; Zhang, Jun; Yue, Zongwei; Song, Jinghui; Zong, Shuai; Liu, Menghao; Stovicek, Olivia; Gao, Yi Qin; Yi, Chengqi

    2016-01-01

    NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)—a preferred substrate—for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction. PMID:27354518

  3. DNA base excision repair nanosystem engineering: model development.

    PubMed

    Sokhansanj, B A

    2005-01-01

    DNA base damage results from a combination of endogenous sources, (normal metabolism, increased metabolism due to obesity, stress from diseases such as arthritis and diabetes, and ischemia) and the environment (ingested toxins, ionizing radiation, etc.). If unrepaired DNA base damage can lead to diminished cell function, and potentially diseases and eventually mutations that lead to cancer. Sophisticated DNA repair mechanisms have evolved in all living cells to preserve the integrity of inherited genetic information and transcriptional control. Understanding a system like DNA repair is greatly enhanced by using engineering methods, in particular modeling interactions and using predictive simulation to analyze the impact of perturbations. We describe the use of such a "nanosystem engineering" approach to analyze the DNA base excision repair pathway in human cells, and use simulation to predict the impact of varying enzyme concentration on DNA repair capacity.

  4. How are base excision DNA repair pathways deployed in vivo?

    PubMed Central

    Thapar, Upasna; Demple, Bruce

    2017-01-01

    Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them. PMID:28357058

  5. How are base excision DNA repair pathways deployed in vivo?

    PubMed

    Thapar, Upasna; Demple, Bruce

    2017-01-01

    Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.

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

    PubMed

    Friedberg, Errol C

    2016-01-01

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

  7. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  8. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair

    PubMed Central

    Çağlayan, Melike; Wilson, Samuel H.

    2015-01-01

    DNA lesions arise from many endogenous and environmental agents, and they promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511

  9. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair

    PubMed Central

    çağlayan, Melike; Wilson, Samuel H.

    2015-01-01

    DNA lesions arise from many endogenous and environmental agents, and they promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5′-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26466358

  10. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair.

    PubMed

    Çağlayan, Melike; Wilson, Samuel H

    2015-11-01

    DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.

  11. Human AP Endonuclease I Stimulates Multiple-Turnover Base Excision by Alkyladenine DNA Glycosylase†

    PubMed Central

    Baldwin, Michael R.; O’Brien, Patrick J.

    2009-01-01

    Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of damaged purine bases from DNA, including hypoxanthine that is formed by the oxidative deamination of adenine. We used steady state, pre-steady state, and single-turnover kinetic assays to show that the multiple-turnover excision of hypoxanthine in vitro is limited by release of the abasic DNA product. This suggests the possibility that the product release step is regulated in vivo by interactions with other base excision repair (BER) proteins. Such coordination of BER activities would protect the abasic DNA repair intermediate and ensure its correct processing. AP endonuclease 1 (APE1) is the predominant enzyme for processing abasic DNA sites in human cells. Therefore, we have investigated the functional effects of added APE1 on the base excision activity of AAG. We find that APE1 stimulates the multiple-turnover excision of hypoxanthine by AAG, but has no effect on single-turnover excision. Since the amino terminus of AAG has been implicated in other protein-protein interactions we also characterize the deletion mutant lacking the first 79 amino acids. We find that APE1 fully stimulates the multiple-turnover glycosylase activity of this mutant, demonstrating that the amino terminus of AAG is not strictly required for this functional interaction. These results are consistent with a model whereby APE1 displaces AAG from the abasic site, thereby coordinating the first two steps of the base excision repair pathway. PMID:19449863

  12. DNA base excision repair of uracil residues in reconstituted nucleosome core particles

    PubMed Central

    Nilsen, Hilde; Lindahl, Tomas; Verreault, Alain

    2002-01-01

    The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase β and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase β on nucleosome cores. PMID:12411511

  13. DNA excision repair at telomeres.

    PubMed

    Jia, Pingping; Her, Chengtao; Chai, Weihang

    2015-12-01

    DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance.

  14. Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair.

    PubMed

    German, Peter; Szaniszlo, Peter; Hajas, Gyorgy; Radak, Zsolt; Bacsi, Attila; Hazra, Tapas K; Hegde, Muralidhar L; Ba, Xueqing; Boldogh, Istvan

    2013-10-01

    Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.

  15. Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase.

    PubMed

    Adhikari, Sanjay; Uren, Aykut; Roy, Rabindra

    2009-10-02

    N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study, we tested the role of excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N(6)-ethenoadenine (varepsilonA) from varepsilonA-containing oligonucleotide (varepsilonA-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (K(D)) towards MPG, but under single-turnover (STO) condition there is apparently no effect on catalytic chemistry; however, the turnover of the enzyme under multiple-turnover (MTO) condition is higher for varepsilonA-DNA than it is for Hx-DNA. Real-time binding experiments by SPR spectroscopy further showed that the dissociation of MPG from its product, AP-site containing DNA, is faster than the overall turnover of either Hx- or varepsilonA-DNA reaction. We thereby conclude that the excised base plays a critical role in product inhibition and, hence, is essential for MPG glycosylase activity. Thus, the results provide the first evidence that the excised base rather than AP-site could be rate-limiting for DNA-glycosylase reactions.

  16. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    PubMed

    Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Sakuma, Tetsushi; Kobayashi, Tomoko; Yamamoto, Takashi; Koyanagi, Yoshio

    2015-01-01

    DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  17. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).

  18. Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair

    PubMed Central

    Crespan, Emmanuele; Furrer, Antonia; Rösinger, Marcel; Bertoletti, Federica; Mentegari, Elisa; Chiapparini, Giulia; Imhof, Ralph; Ziegler, Nathalie; Sturla, Shana J.; Hübscher, Ulrich; van Loon, Barbara; Maga, Giovanni

    2016-01-01

    Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. PMID:26917111

  19. The role of DNA base excision repair in brain homeostasis and disease.

    PubMed

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah; Bohr, Vilhelm A

    2015-08-01

    Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of proteins required for BER or proteins that regulate BER have been consistently associated with neurological dysfunction and disease in humans. Recent studies suggest that DNA lesions in the nuclear and mitochondrial compartments and the cellular response to those lesions have a profound effect on cellular energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease.

  20. Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1.

    PubMed

    Córdoba-Cañero, Dolores; Roldán-Arjona, Teresa; Ariza, Rafael R

    2011-11-01

    Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.

  1. Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA.

    PubMed

    Shafirovich, Vladimir; Kropachev, Konstantin; Anderson, Thomas; Liu, Zhi; Kolbanovskiy, Marina; Martin, Brooke D; Sugden, Kent; Shim, Yoonjung; Chen, Xuejing; Min, Jung-Hyun; Geacintov, Nicholas E

    2016-03-04

    The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC(-/-) fibroblast cell extracts. However, normal NER activity appeared when the XPC(-/-) cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1(-/-) relative to NEIL1(+/+) extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions.

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

    PubMed

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

    2016-06-21

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

  3. Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair

    PubMed Central

    Cortellino, Salvatore; Xu, Jinfei; Sannai, Mara; Moore, Robert; Caretti, Elena; Cigliano, Antonio; Le Coz, Madeleine; Devarajan, Karthik; Wessels, Andy; Soprano, Dianne; Abramowitz, Lara K.; Bartolomei, Marisa S.; Rambow, Florian; Bassi, Maria Rosaria; Bruno, Tiziana; Fanciulli, Maurizio; Renner, Catherine; Klein-Szanto, Andres J.; Matsumoto, Yoshihiro; Kobi, Dominique; Davidson, Irwin; Alberti, Christophe; Larue, Lionel; Bellacosa, Alfonso

    2011-01-01

    Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair. PMID:21722948

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

    PubMed

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

    2016-12-20

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

  5. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

    NASA Astrophysics Data System (ADS)

    Mullins, Elwood A.; Shi, Rongxin; Parsons, Zachary D.; Yuen, Philip K.; David, Sheila S.; Igarashi, Yasuhiro; Eichman, Brandt F.

    2015-11-01

    Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

  6. Oxidative DNA damage background estimated by a system model of base excision repair

    SciTech Connect

    Sokhansanj, B A; Wilson, III, D M

    2004-05-13

    Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.

  7. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  8. Oxidative DNA damage background estimated by a system model of base excision repair.

    PubMed

    Sokhansanj, Bahrad A; Wilson, David M

    2004-08-01

    Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level, based on measuring 8-oxoguanine lesions as a biomarker, have led to estimates that vary over three to four orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our findings show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.

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

    PubMed

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

    2015-08-01

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

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

    PubMed

    Grasso, Stefano; Tell, Gianluca

    2014-09-01

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

  11. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  12. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  13. On-bead fluorescent DNA nanoprobes to analyze base excision repair activities.

    PubMed

    Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier

    2014-02-17

    DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes' activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL(-1) and 50 μg mL(-1) of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair activities.

  14. Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

    PubMed Central

    Fleming, Aaron M.; Ding, Yun; Burrows, Cynthia J.

    2017-01-01

    Reactive oxygen species (ROS) have emerged as important cellular-signaling agents for cellular survival. Herein, we demonstrate that ROS-mediated oxidation of DNA to yield 8-oxo-7,8-dihydroguanine (OG) in gene promoters is a signaling agent for gene activation. Enhanced gene expression occurs when OG is formed in guanine-rich, potential G-quadruplex–forming sequences (PQS) in promoter-coding strands, initiating base excision repair (BER) by 8-oxoguanine DNA glycosylase (OGG1), yielding an abasic site (AP). The AP enables melting of the duplex to unmask the PQS, adopting a G-quadruplex fold in which apurinic/apyrimidinic endonuclease 1 (APE1) binds, but inefficiently cleaves, the AP for activation of vascular endothelial growth factor (VEGF) or endonuclease III-like protein 1 (NTHL1) genes. These details were mapped via synthesis of OG and AP analogs at single-nucleotide precision within the promoter of a luciferase reporter system. The reporters were analyzed in human and mouse cells while selectively knocking out or down critical BER proteins to identify the impact on luciferase expression. Identification of the oxidatively modified DNA base OG to guide BER activity in a gene promoter and impact cellular phenotype ascribes an epigenetic role to OG. PMID:28143930

  15. Base excision DNA repair levels in mitochondrial lysates of Alzheimer's disease.

    PubMed

    Canugovi, Chandrika; Shamanna, Raghavendra A; Croteau, Deborah L; Bohr, Vilhelm A

    2014-06-01

    Alzheimer's disease (AD) is a senile dementia with increased incidence in older subjects (age >65 years). One of the earliest markers of AD is oxidative DNA damage. Recently, it has been reported that preclinical AD patient brains show elevated levels of oxidative damage in both nuclear and mitochondrial nucleic acids. Moreover, different oxidative lesions in mitochondrial DNA are between 5- and 10-fold higher than in nuclear DNA in both control and AD postmortem brains. We previously showed that there is a significant loss of base excision repair (BER) components in whole tissue extracts of AD and mild cognitive impairment subjects relative to matched control subjects. However, comprehensive analysis of specific steps in BER levels in mitochondrial extracts of AD patient brains is not available. In this study, we mainly investigated various components of BER in mitochondrial extracts of AD and matched control postmortem brain samples. We found that the 5-hydroxyuracil incision and ligase activities are significantly lower in AD brains, whereas the uracil incision, abasic site cleavage, and deoxyribonucleotide triphosphate incorporation activities are normal in these samples.

  16. A quantitative model of human DNA base excision repair. I. Mechanistic insights.

    PubMed

    Sokhansanj, Bahrad A; Rodrigue, Garry R; Fitch, J Patrick; Wilson, David M

    2002-04-15

    Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts considerably less pathway throughput than observed in experimental in vitro assays. This finding, in combination with the effects of pathway cooperativity on model throughput, supports the hypothesis of cooperation during abasic site repair and between the apurinic/apyrimidinic (AP) endonuclease, Ape1, and the 8-oxoguanine DNA glycosylase, Ogg1. The quantitative model also predicts that for 8-oxoguanine and hydrolytic AP site damage, short-patch Polbeta-mediated BER dominates, with minimal switching to the long-patch subpathway. Sensitivity analysis of the model indicates that the Polbeta-catalyzed reactions have the most control over pathway throughput, although other BER reactions contribute to pathway efficiency as well. The studies within represent a first step in a developing effort to create a predictive model for BER cellular capacity.

  17. A quantitative model of human DNA base excision repair. I. mechanistic insights

    PubMed Central

    Sokhansanj, Bahrad A.; Rodrigue, Garry R.; Fitch, J. Patrick; Wilson, David M.

    2002-01-01

    Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts considerably less pathway throughput than observed in experimental in vitro assays. This finding, in combination with the effects of pathway cooperativity on model throughput, supports the hypothesis of cooperation during abasic site repair and between the apurinic/apyrimidinic (AP) endonuclease, Ape1, and the 8-oxoguanine DNA glycosylase, Ogg1. The quantitative model also predicts that for 8-oxoguanine and hydrolytic AP site damage, short-patch Polβ-mediated BER dominates, with minimal switching to the long-patch subpathway. Sensitivity analysis of the model indicates that the Polβ-catalyzed reactions have the most control over pathway throughput, although other BER reactions contribute to pathway efficiency as well. The studies within represent a first step in a developing effort to create a predictive model for BER cellular capacity. PMID:11937636

  18. Minimal role of base excision repair in TET-induced global DNA demethylation in HEK293T cells

    PubMed Central

    Jin, Chunlei; Qin, Taichun; Barton, Michelle Craig; Jelinek, Jaroslav; Issa, Jean-Pierre J

    2015-01-01

    Oxidation of 5-methylcytosine by TET family proteins can induce DNA replication-dependent (passive) DNA demethylation and base excision repair (BER)-based (active) DNA demethylation. The balance of active vs. passive TET-induced demethylation remains incompletely determined. In the context of large scale DNA demethylation, active demethylation may require massive induction of the DNA repair machinery and thus compromise genome stability. To study this issue, we constructed a tetracycline-controlled TET-induced global DNA demethylation system in HEK293T cells. Upon TET overexpression, we observed induction of DNA damage and activation of a DNA damage response; however, BER genes are not upregulated to promote DNA repair. Depletion of TDG (thymine DNA glycosylase) or APEX1 (apurinic/apyrimidinic endonuclease 1), two key BER enzymes, enhances rather than impairs global DNA demethylation, which can be explained by stimulated proliferation. By contrast, growth arrest dramatically blocks TET-induced global DNA demethylation. Thus, in the context of TET-induction in HEK293T cells, the DNA replication-dependent passive mechanism functions as the predominant pathway for global DNA demethylation. In the same context, BER-based active demethylation is markedly restricted by limited BER upregulation, thus potentially preventing a disastrous DNA damage response to extensive active DNA demethylation. PMID:26440216

  19. The Potential Role of 8-Oxoguanine DNA Glycosylase-Driven DNA Base Excision Repair in Exercise-Induced Asthma

    PubMed Central

    Belanger, KarryAnne K.; Ameredes, Bill T.; Boldogh, Istvan

    2016-01-01

    Asthma is characterized by reversible airway narrowing, shortness of breath, wheezing, coughing, and other symptoms driven by chronic inflammatory processes, commonly triggered by allergens. In 90% of asthmatics, most of these symptoms can also be triggered by intense physical activities and severely exacerbated by environmental factors. This condition is known as exercise-induced asthma (EIA). Current theories explaining EIA pathogenesis involve osmotic and/or thermal alterations in the airways caused by changes in respiratory airflow during exercise. These changes, along with existing airway inflammatory conditions, are associated with increased cellular levels of reactive oxygen species (ROS) affecting important biomolecules including DNA, although the underlying molecular mechanisms have not been completely elucidated. One of the most abundant oxidative DNA lesions is 8-oxoguanine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase 1 (OGG1) during the base excision repair (BER) pathway. Whole-genome expression analyses suggest a cellular response to OGG1-BER, involving genes that may have a role in the pathophysiology of EIA leading to mast cell degranulation, airway hyperresponsiveness, and bronchoconstriction. Accordingly, this review discusses a potential new hypothesis in which OGG1-BER-induced gene expression is associated with EIA symptoms. PMID:27524866

  20. Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages*

    PubMed Central

    Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.

    2014-01-01

    Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506

  1. Inroads into base excision repair II. The discovery of DNA glycosylases. "An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues," Proc. Nat. Acad. Sci. USA, 1974.

    PubMed

    Friedberg, Errol C; Lindahl, Tomas

    2004-11-02

    The discovery of a DNA glycosylase that specifically removes uracil from DNA, opened the door for uncovering a large class of such enzymes that are fundamental to the process of base excision repair of DNA.

  2. Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage.

    PubMed

    Sokhansanj, Bahrad A; Wilson, David M

    2006-05-01

    Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage.

  3. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

    SciTech Connect

    Wang, Ping; Guliaev, Anton B.; Hang, Bo

    2006-02-28

    Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  4. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    PubMed

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion.

  5. The nucleoid-associated protein HU enhances 8-oxoguanine base excision by the formamidopyrimidine-DNA glycosylase.

    PubMed

    Le Meur, Rémy; Culard, Françoise; Nadan, Virginie; Goffinont, Stéphane; Coste, Franck; Guerin, Martine; Loth, Karine; Landon, Céline; Castaing, Bertrand

    2015-10-01

    The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.

  6. Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

    PubMed

    Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

    2014-04-01

    DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

  7. An alternative eukaryotic DNA excision repair pathway.

    PubMed Central

    Freyer, G A; Davey, S; Ferrer, J V; Martin, A M; Beach, D; Doetsch, P W

    1995-01-01

    DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe. PMID:7623848

  8. 3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

    PubMed Central

    Schuermann, David; Scheidegger, Simon P.; Weber, Alain R.; Bjørås, Magnar; Leumann, Christian J.; Schär, Primo

    2016-01-01

    Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases. PMID:26733580

  9. 3CAPS - a structural AP-site analogue as a tool to investigate DNA base excision repair.

    PubMed

    Schuermann, David; Scheidegger, Simon P; Weber, Alain R; Bjørås, Magnar; Leumann, Christian J; Schär, Primo

    2016-03-18

    Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3'-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5'-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

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

    PubMed Central

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

    2015-01-01

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 hours. In cultures where APE1 expression was reduced by ~80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Reduction in APE1 expression also altered the expression of the NER proteins RPA70 and XPA in sensory neuronal cultures. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226+177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons. PMID:26164266

  11. Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite

    SciTech Connect

    Sykora, Peter; Snow, Elizabeth T.

    2008-05-01

    Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase {beta} (Pol {beta}) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol {beta} and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 {mu}M. However, at lower doses Pol {beta} mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol {beta} was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis.

  12. A Catalytic Role for C-H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD.

    PubMed

    Parsons, Zachary D; Bland, Joshua M; Mullins, Elwood A; Eichman, Brandt F

    2016-09-14

    DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually take place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the C-H/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair.

  13. DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA

    PubMed Central

    Hollis, Thomas; Ichikawa, Yoshitaka; Ellenberger, Tom

    2000-01-01

    The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 Å crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1–azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1–azaribose in the enzyme active site suggests an SN1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner. PMID:10675345

  14. Both base excision repair and O6-methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis

    PubMed Central

    Wirtz, Stefan; Nagel, Georg; Eshkind, Leonid; Neurath, Markus F.; Samson, Leona D.; Kaina, Bernd

    2010-01-01

    Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O6-methylguanine (O6MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O6-methylguanine-DNA methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O6MeG is well established as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3-methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced colon carcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O6MeG reversal by Mgmt are required for protection against alkylation-induced colon carcinogenesis. Further, the data indicate that non-repaired N-methylpurines are not only pre-toxic but also pre-carcinogenic DNA lesions. PMID:20732909

  15. RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

    PubMed

    Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

    2010-06-04

    The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.

  16. A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

    PubMed Central

    Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei; Guikema, Jeroen E. J.; Schrader, Carol E.; Strauss, Phyllis R.

    2011-01-01

    DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex+/− mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes. PMID:21172930

  17. Base excision repair of oxidative DNA damage coupled with removal of a CAG repeat hairpin attenuates trinucleotide repeat expansion.

    PubMed

    Xu, Meng; Lai, Yanhao; Torner, Justin; Zhang, Yanbin; Zhang, Zunzhen; Liu, Yuan

    2014-04-01

    Trinucleotide repeat (TNR) expansion is responsible for numerous human neurodegenerative diseases. However, the underlying mechanisms remain unclear. Recent studies have shown that DNA base excision repair (BER) can mediate TNR expansion and deletion by removing base lesions in different locations of a TNR tract, indicating that BER can promote or prevent TNR expansion in a damage location-dependent manner. In this study, we provide the first evidence that the repair of a DNA base lesion located in the loop region of a CAG repeat hairpin can remove the hairpin, attenuating repeat expansion. We found that an 8-oxoguanine located in the loop region of CAG hairpins of varying sizes was removed by OGG1 leaving an abasic site that was subsequently 5'-incised by AP endonuclease 1, introducing a single-strand breakage in the hairpin loop. This converted the hairpin into a double-flap intermediate with a 5'- and 3'-flap that was cleaved by flap endonuclease 1 and a 3'-5' endonuclease Mus81/Eme1, resulting in complete or partial removal of the CAG hairpin. This further resulted in prevention and attenuation of repeat expansion. Our results demonstrate that TNR expansion can be prevented via BER in hairpin loops that is coupled with the removal of TNR hairpins.

  18. Interplay between base excision repair activity and toxicity of 3-methyladenine DNA glycosylases in an E. coli complementation system.

    PubMed

    Troll, Christopher J; Adhikary, Suraj; Cueff, Marie; Mitra, Ileena; Eichman, Brandt F; Camps, Manel

    2014-01-01

    DNA glycosylases carry out the first step of base excision repair by removing damaged bases from DNA. The N3-methyladenine (3MeA) DNA glycosylases specialize in alkylation repair and are either constitutively expressed or induced by exposure to alkylating agents. To study the functional and evolutionary significance of constitutive versus inducible expression, we expressed two closely related yeast 3MeA DNA glycosylases - inducible Saccharomyces cerevisiae MAG and constitutive S. pombe Mag1 - in a glycosylase-deficient Escherichia coli strain. In both cases, constitutive expression conferred resistance to alkylating agent exposure. However, in the absence of exogenous alkylation, high levels of expression of both glycosylases were deleterious. We attribute this toxicity to excessive glycosylase activity, since suppressing spMag1 expression correlated with improved growth in liquid culture, and spMag1 mutants exhibiting decreased glycosylase activity showed improved growth and viability. Selection of a random spMag1 mutant library for increased survival in the presence of exogenous alkylation resulted in the selection of hypomorphic mutants, providing evidence for the presence of a genetic barrier to the evolution of enhanced glycosylase activity when constitutively expressed. We also show that low levels of 3MeA glycosylase expression improve fitness in our glycosylase-deficient host, implying that 3MeA glycosylase activity is likely necessary for repair of endogenous lesions. These findings suggest that 3MeA glycosylase activity is evolutionarily conserved for repair of endogenously produced alkyl lesions, and that inducible expression represents a common strategy to rectify deleterious effects of excessive 3MeA activity in the absence of exogenous alkylation challenge.

  19. The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

    PubMed

    Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2012-11-01

    Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.

  20. In vivo treatment with aflatoxin B1 increases DNA oxidation, base excision repair activity and 8-oxoguanine DNA glycosylase 1 levels in mouse lung.

    PubMed

    Guindon-Kezis, Katherine A; Mulder, Jeanne E; Massey, Thomas E

    2014-07-03

    Carcinogenicity of the mycotoxin aflatoxin B1 (AFB1), which is produced by Aspergillus fungi, is associated with bioactivation of AFB1 to AFB1-8,9-exo-epoxide and formation of DNA adducts. However, AFB1 also causes 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in mouse lung DNA, suggesting that oxidative DNA damage may also contribute to AFB1 carcinogenicity. The oxidative DNA damage 5-hydroxy-2'-deoxycytidine (5-OHdC) may also contribute to AFB1 carcinogenicity. The objective of the present study was to determine the effect of treatment of mice with AFB1 on pulmonary and hepatic: 8-OHdG and 5-OHdC levels; base excision repair (BER, which repairs oxidative DNA damage) activities; and on levels of 8-oxoguanine DNA glycosylase (OGG1, the rate-limiting enzyme in the BER of 8-OHdG). Female A/J mice were treated with vehicle (dimethyl sulfoxide) or 50 mg/kg AFB1 ip. Oxidative DNA damage was measured using HPLC with electrochemical detection, BER activity was assessed using an in vitro assay that employs a substrate plasmid DNA with 8-OHdG lesions, and OGG1 protein levels were determined by immunoblotting. Two hours post treatment, AFB1 increased 8-OHdG levels in mouse lung DNA by approximately 69% relative to control (p<0.05), but did not alter 8-OHdG levels in liver or 5-OHdC levels in lung or liver (p>0.05). AFB1 treatment also increased BER activity in mouse lung by approximately 87% (p<0.05) but did not affect hepatic BER activity (p>0.05). Levels of OGG1 immunoreactive protein were increased in both lung (20%) and liver (60%) (p<0.05). These results are consistent with oxidative DNA damage contributing to the carcinogenicity of AFB1 in this model.

  1. Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

    PubMed Central

    Kelley, Mark R.; Jiang, Yanlin; Guo, Chunlu; Reed, April; Meng, Hongdi; Vasko, Michael R.

    2014-01-01

    Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE

  2. A multiplex assay based on encoded microbeads conjugated to DNA NanoBeacons to monitor base excision repair activities by flow cytometry.

    PubMed

    Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier

    2014-08-15

    We reported here a new assay to detect base excision repair activities from purified enzymes, as well as in cell-free extracts. The multiplex format rests upon the encoding of magnetic beads with the fluorophore Alexa 488, thanks to a fast and original procedure. Fluorescently encoded microbeads are subsequently functionalized by lesion-containing DNA NanoBeacons labeled with the fluorophore/quencher pair Cyanine 5/BHQ2. Probes cleavage, induced by targeted enzymes leads to Cyanine 5 signal enhancement, which is finally quantified by flow cytometry. The multiplex assay was applied to the detection of restriction enzymes activities as well as base excision repair processes. Each test requires only 500fmol of DNA substrate, which constitutes great sensitivity compared to other BER functional assays. The present biosensor is able to detect both uracil DNA N-glycosylase (UNG) and AP-endonuclease 1 (APE1) within few nanograms of nuclear extract. Additionally, we demonstrated that the corresponding assay has potential application in DNA repair inhibitor search. Finally, the current multiplexed tool shows several advantages in comparison to other functional BER assays with no need of electrophoretic separation, straightforward, easy and reproducible functionalization of encoded microbeads and a high stability of DNA probes in cell-free extracts.

  3. Distant neighbor base sequence context effects in human nucleotide excision repair of a benzo[a]pyrene-derived DNA lesion.

    PubMed

    Cai, Yuqin; Kropachev, Konstantin; Xu, Rong; Tang, Yijin; Kolbanovskii, Marina; Kolbanovskii, Alexander; Amin, Shantu; Patel, Dinshaw J; Broyde, Suse; Geacintov, Nicholas E

    2010-06-11

    The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N(2)-dG (G*) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5'-C-C-A-T-C-G*-C-T-A-C-C-3' (CG*C-I), and 5'-C-A-C3-A4-C5-G*-C-A-C-A-C-3' (CG*C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(+/-0.2)-fold greater in the case of the CG*C-II than the CG*C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG*C-II duplex is more bent than the CG*C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG*C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG*C-II than in CG*C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG*C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N(2)-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.

  4. The nucleotide sequence, DNA damage location, and protein stoichiometry influence the base excision repair outcome at CAG/CTG repeats.

    PubMed

    Goula, Agathi-Vasiliki; Pearson, Christopher E; Della Maria, Julie; Trottier, Yvon; Tomkinson, Alan E; Wilson, David M; Merienne, Karine

    2012-05-08

    Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington's disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5' end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3' end of the repeats or within control sequences. Moreover, repair of lesions at the 5' end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability.

  5. A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair.

    PubMed

    Wei, Shanqiao; Shalhout, Sophia; Ahn, Young-Hoon; Bhagwat, Ashok S

    2015-03-01

    A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

  6. Characterizing Requirements for Small Ubiquitin-like Modifier (SUMO) Modification and Binding on Base Excision Repair Activity of Thymine-DNA Glycosylase in Vivo.

    PubMed

    McLaughlin, Dylan; Coey, Christopher T; Yang, Wei-Chih; Drohat, Alexander C; Matunis, Michael J

    2016-04-22

    Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.

  7. Molecular Cloning and 3D Structure Modeling of APEX1, DNA Base Excision Repair Enzyme from the Camel, Camelus dromedarius

    PubMed Central

    Ataya, Farid Shokry; Fouad, Dalia; Malik, Ajamaluddin; Saeed, Hesham Mahmoud

    2012-01-01

    The domesticated one-humped camel, Camelus dromedarius, is one of the most important animals in the Arabian Desert. It is exposed most of its life to both intrinsic and extrinsic genotoxic factors that are known to cause gross DNA alterations in many organisms. Ionic radiation and sunlight are known producers of Reactive Oxygen Species (ROS), one of the causes for DNA lesions. The damaged DNA is repaired by many enzymes, among of them Base Excision Repair enzymes, producing the highly mutagenic apurinic/apyrimidinicsites (AP sites). Therefore, recognition of AP sites is fundamental to cell/organism survival. In the present work, the full coding sequence of a putative cAPEX1 gene was amplified for the first time from C. dromedarius by RT-PCR and cloned (NCBI accession number are HM209828 and ADJ96599 for nucleotides and amino acids, respectively). cDNA sequencing was deduced to be 1041 nucleotides, of which 954 nucleotides encode a protein of 318 amino acids, similar to the coding region of the APEX1 gene and the protein from many other species. The calculated molecular weight and isoelectric point of cAPEX1 using Bioinformatics tools was 35.5 kDa and 8.11, respectively. The relative expressions of cAPEX1 in camel kidney, spleen, lung and testis were examined using qPCR and compared with that of the liver using a 18S ribosomal subunit as endogenous control. The highest level of cAPEX1 transcript was found in the testis; 325% higher than the liver, followed by spleen (87%), kidney (20%) and lung (5%), respectively. The cAPEX1 is 94%–97% similar to their mammalian counterparts. Phylogenetic analysis revealed that cAPEX1 is grouped together with that of S. scrofa. The predicted 3D structure of cAPEX1 has similar folds and topology with the human (hAPEX1). The root-mean-square deviation (rmsd) between cAPEX1 and hAPEX1 was 0.582 and the Q-score was 0.939. PMID:22942721

  8. DNA excision repair in permeable human fibroblasts

    SciTech Connect

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

    1983-01-01

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

  9. Guanine oxidation product 5-carboxamido-5-formamido-2-iminohydantoin induces mutations when bypassed by DNA polymerases and is a substrate for base excision repair.

    PubMed

    Alshykhly, Omar R; Fleming, Aaron M; Burrows, Cynthia J

    2015-09-21

    Guanine (G) is a target for oxidation by reactive oxygen species in DNA, RNA, and the nucleotide pool. Damage to DNA yields products with alternative properties toward DNA processing enzymes compared to those of the parent nucleotide. A new lesion, 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), bearing a stereocenter in the base was recently identified from the oxidation of G. DNA polymerase and base excision repair processing of this new lesion has now been evaluated. Single nucleotide insertion opposite (S)-2Ih and (R)-2Ih in the template strand catalyzed by the DNA polymerases Klenow fragment exo(-), DPO4, and Hemo KlenTaq demonstrates these lesions to cause point mutations. Specifically, they promote 3-fold more G·C → C·G transversion mutations than G·C → T·A, and (S)-2Ih was 2-fold more blocking for polymerase bypass than (R)-2Ih. Both diastereomer lesions were found to be substrates for the DNA glycosylases NEIL1 and Fpg, and poorly excised by endonuclease III (Nth). The activity was independent of the base pair partner. Thermal melting, CD spectroscopy, and density functional theory geometric optimization calculations were conducted to provide insight into these polymerase and DNA glycosylase studies. These results identify that formation of the 2Ih lesions in a cell would be mutagenic in the event that they were not properly repaired.

  10. Recombinant methods for screening human DNA excision repair proficiency

    SciTech Connect

    Athas, W.F.

    1988-01-01

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

  11. Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways.

    PubMed

    Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T; Gasparutto, Didier; Geacintov, Nicholas E; Saparbaev, Murat

    2015-06-05

    Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.

  12. Variation in Base Excision Repair Capacity

    PubMed Central

    Wilson, David M.; Kim, Daemyung; Berquist, Brian R.; Sigurdson, Alice J.

    2010-01-01

    The major DNA repair pathway for coping with spontaneous forms of DNA damage, such as natural hydrolytic products or oxidative lesions, is base excision repair (BER). In particular, BER processes mutagenic and cytotoxic DNA lesions such as non-bulky base modifications, abasic sites, and a range of chemically distinct single-strand breaks. Defects in BER have been linked to cancer predisposition, neurodegenerative disorders, and immunodeficiency. Recent data indicate a large degree of sequence variability in DNA repair genes and several studies have associated BER gene polymorphisms with disease risk, including cancer of several sites. The intent of this review is to describe the range of BER capacity among individuals and the functional consequences of BER genetic variants. We also discuss studies that associate BER deficiency with disease risk and the current state of BER capacity measurement assays. PMID:21167187

  13. Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism.

    PubMed

    Page, Melissa M; Salmon, Adam B; Leiser, Scott F; Robb, Ellen L; Brown, Melanie F; Miller, Richard A; Stuart, Jeffrey A

    2009-04-15

    Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O(2); the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O(2), was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts.

  14. Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver.

    PubMed

    Rusyn, Ivan; Asakura, Shoji; Li, Yutai; Kosyk, Oksana; Koc, Hasan; Nakamura, Jun; Upton, Patricia B; Swenberg, James A

    2005-09-28

    Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8

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

    PubMed Central

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

    2014-01-01

    Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  16. Nonuniform distribution of excision repair synthesis in nucleosome core DNA

    SciTech Connect

    Lan, S.Y.; Smerdon, M.J.

    1985-12-17

    We have studied the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. The differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to map the distribution of repair synthesis in these regions. Results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. The 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only internal locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.

  17. Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate: New Evidence on the Role of PARP-1 in DNA Repair

    SciTech Connect

    Lavrik, Olga I.; Prasad, Rajendra; Sobol, Robert W.; Horton, Julie K.; Ackerman, Eric J. ); Wilson, Samuel H.

    2001-07-06

    To examine mammalian base excision repair (BER) enzymes interacting with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast (MEF) crude extract. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by AP endonuclease creating a nick with 3' hydroxyl and 5' reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was introduced at the 3' hydroxyl group. With near UV-light exposure (312nm) of the extract-probe mixture, only six proteins were strongly labeled, including poly (ADP-ribose) polymerase (PARP-1) and the well-known BER participants flap endonuclease (FEN-1), DNA polymerase b (b-pol), and AP endonuclease (APE). The amount of probe crosslinked to PARP-1 was greater than that crosslinked to the other proteins. The specificity of PARP-1 labeling was examined by competition experiments involving various oligonucleotide competitors; competition of labeling by the probe was much greater for the BER intermediates tested than for normal double-stranded DNA. The specificity of PARP-1 labeling also was examined using DNA probes with alternate structures; PARP-1 labeling was stronger with a DNA oligomer representing a BER intermediate than with a molecule representing a nick in double-stranded DNA. These results identifying interaction of PARP-1 with a BER intermediate are discussed in light of PARP-1's role in mammalian BER.

  18. DNA-protein crosslinks processed by nucleotide excision repair and homologous recombination with base and strand preference in E. coli model system.

    PubMed

    Fang, Qingming

    2013-01-01

    Bis-electrophiles including dibromoethane and epibromohydrin can react with O(6)-alkylguanine-DNA alkyltransferase (AGT) and form AGT-DNA crosslinks in vitro and in vivo. The presence of human AGT (hAGT) paradoxically increases the mutagenicity and cytotoxicity of bis-electrophiles in cells. Here we establish a bacterial system to study the repair mechanism and cellular responses to DNA-protein crosslinks (DPCs) in vivo. Results show that both nucleotide excision repair (NER) and homologous recombination (HR) pathways can process hAGT-DNA crosslinks with HR playing a dominant role. Mutation spectra show that HR has no strand preference but NER favors processing of the DPCs in the transcribed strand; UvrA, UvrB and Mfd can interfere with small size DPCs but only UvrA can interfere with large size DPCs in the transcribed strand processed by HR. Further, we found that DPCs at TA deoxynucleotide sites are very inefficiently processed by NER and the presence of NER can interfere with these DNA lesions processed by HR. These data indicate that NER and HR can process DPCs cooperatively and competitively and NER processes DPCs with base and strand preference. Therefore, the formation of hAGT-DNA crosslinks can be a plausible and specific system to study the repair mechanism and effects of DPCs precisely in vivo.

  19. Nucleotide excision repair of DNA: The very early history.

    PubMed

    Friedberg, Errol C

    2011-07-15

    This article, taken largely from the book Correcting the Blueprint of Life: An Historical Account of the Discovery of DNA Repair Mechanisms, summarizes the very early history of the discovery of nucleotide excision repair.

  20. Coordination of Steps in Single-nucleotide Base Excision Repair Mediated by Apurinic/Apyrimidinic Endonuclease 1 and DNA Polymerase β*

    PubMed Central

    Liu, Yuan; Prasad, Rajendra; Beard, William A.; Kedar, Padmini S.; Hou, Esther W.; Shock, David D.; Wilson, Samuel H.

    2008-01-01

    The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase β (Pol β). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE·Pol β·DNA that formed specifically at BER intermediates containing a 5′-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol β gap-filling and much stronger 5′-deoxyribose phosphate lyase activities than was observed with the Pol β·DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol β, although coordination also may be facilitated by APE·Pol β·DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair. PMID:17355977

  1. Base excision repair capacity in informing healthspan

    PubMed Central

    Brenerman, Boris M.; Illuzzi, Jennifer L.; Wilson, David M.

    2014-01-01

    Base excision repair (BER) is a frontline defense mechanism for dealing with many common forms of endogenous DNA damage, several of which can drive mutagenic or cell death outcomes. The pathway engages proteins such as glycosylases, abasic endonucleases, polymerases and ligases to remove substrate modifications from DNA and restore the genome back to its original state. Inherited mutations in genes related to BER can give rise to disorders involving cancer, immunodeficiency and neurodegeneration. Studies employing genetically defined heterozygous (haploinsufficient) mouse models indicate that partial reduction in BER capacity can increase vulnerability to both spontaneous and exposure-dependent pathologies. In humans, measurement of BER variation has been imperfect to this point, yet tools to assess BER in epidemiological surveys are steadily evolving. We provide herein an overview of the BER pathway and discuss the current efforts toward defining the relationship of BER defects with disease susceptibility. PMID:25355293

  2. Tumor-selective use of DNA base excision repair inhibition in pancreatic cancer using the NQO1 bioactivatable drug, β-lapachone

    PubMed Central

    Chakrabarti, Gaurab; Silvers, Molly A.; Ilcheva, Mariya; Liu, Yuliang; Moore, Zachary R.; Luo, Xiuquan; Gao, Jinming; Anderson, Glenda; Liu, Lili; Sarode, Venetia; Gerber, David E.; Burma, Sandeep; DeBerardinis, Ralph J.; Gerson, Stanton L.; Boothman, David A.

    2015-01-01

    Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The NAD(P)H:Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD+/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures. Significance: Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer. PMID:26602448

  3. Increased oxidative DNA damage and decreased expression of base excision repair proteins in airway epithelial cells of women who cook with biomass fuels.

    PubMed

    Mukherjee, Bidisha; Bindhani, Banani; Saha, Hirak; Ray, Manas Ranjan

    2014-09-01

    To investigate whether biomass burning causes oxidative DNA damage and alters the expression of DNA base excision repair (BER) proteins in airway cells, sputum samples were collected from 80 premenopausal rural biomass-users and 70 age-matched control women who cooked with liquefied petroleum gas. Compared with control the airway cells of biomass-users showed increased DNA damage in alkaline comet assay. Biomass-users showed higher percentage of cells expressing oxidative DNA damage marker 8-oxoguanine and lower percentages of BER proteins OGG1 and APE1 by immunocytochemical staining. Reactive oxygen species (ROS) generation was doubled and level of superoxide dismutase was depleted significantly among biomass-users. The concentrations of particulate matters were higher in biomass-using households which positively correlated with ROS generation and negatively with BER proteins expressions. ROS generation was positively correlated with 8-oxoguanine and negatively with BER proteins suggesting cooking with biomass is a risk for genotoxicity among rural women in their child-bearing age.

  4. Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    PubMed Central

    de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel

    2016-01-01

    Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709

  5. Nucleotide exchange and excision technology DNA shuffling and directed evolution.

    PubMed

    Speck, Janina; Stebel, Sabine C; Arndt, Katja M; Müller, Kristian M

    2011-01-01

    Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.

  6. Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA.

    PubMed

    Bartels, Phillip L; Zhou, Andy; Arnold, Anna R; Nuñez, Nicole N; Crespilho, Frank N; David, Sheila S; Barton, Jacqueline K

    2017-03-02

    Escherichia coli endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S] clusters and that serve to maintain the integrity of the genome after oxidative stress. Electrochemical studies on highly oriented pyrolytic graphite (HOPG) revealed that DNA binding by EndoIII leads to a large negative shift in the midpoint potential of the cluster, consistent with stabilization of the oxidized [4Fe4S](3+) form. However, the smooth, hydrophobic HOPG surface is nonideal for working with proteins in the absence of DNA. In this work, we use thin film voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the cluster, all show DNA-free potentials within error of wild type; similarly, the presence of negatively charged poly-l-glutamate does not lead to a significant potential shift. Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster, helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have similar DNA-bound potentials.

  7. Alcohol-induced one-carbon metabolism impairment promotes dysfunction of DNA base excision repair in adult brain.

    PubMed

    Fowler, Anna-Kate; Hewetson, Aveline; Agrawal, Rajiv G; Dagda, Marisela; Dagda, Raul; Moaddel, Ruin; Balbo, Silvia; Sanghvi, Mitesh; Chen, Yukun; Hogue, Ryan J; Bergeson, Susan E; Henderson, George I; Kruman, Inna I

    2012-12-21

    The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

  8. Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain*

    PubMed Central

    Fowler, Anna-Kate; Hewetson, Aveline; Agrawal, Rajiv G.; Dagda, Marisela; Dagda, Raul; Moaddel, Ruin; Balbo, Silvia; Sanghvi, Mitesh; Chen, Yukun; Hogue, Ryan J.; Bergeson, Susan E.; Henderson, George I.; Kruman, Inna I.

    2012-01-01

    The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain. PMID:23118224

  9. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    PubMed

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón Y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; Swe-Brca; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I; Beattie, Mary S; Domchek, Susan M; Nathanson, Katherine; Rebbeck, Timothy R; Arun, Banu K; Karlan, Beth Y; Walsh, Christine; Lester, Jenny; John, Esther M; Whittemore, Alice S; Daly, Mary B; Southey, Melissa; Hopper, John; Terry, Mary B; Buys, Saundra S; Janavicius, Ramunas; Dorfling, Cecilia M; van Rensburg, Elizabeth J; Steele, Linda; Neuhausen, Susan L; Ding, Yuan Chun; Hansen, Thomas V O; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A; van Os, Theo A M; van der Kolk, Lizet; de Lange, J L; Meijers-Heijboer, Hanne E J; van der Hout, A H; van Asperen, Christi J; Gómez Garcia, Encarna B; Hoogerbrugge, Nicoline; Collée, J Margriet; van Deurzen, Carolien H M; van der Luijt, Rob B; Devilee, Peter; Hebon; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R; Healey, Sue; Investigators, Kconfab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M; Greene, Mark H; Mai, Phuong L; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F; Offit, Kenneth; Couch, Fergus J; Chenevix-Trench, Georgia; Antoniou, Antonis C; Benitez, Javier

    2014-04-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  10. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    PubMed Central

    Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; SWE-BRCA; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I.; Beattie, Mary S.; Domchek, Susan M.; Nathanson, Katherine; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; John, Esther M.; Whittemore, Alice S.; Daly, Mary B.; Southey, Melissa; Hopper, John; Terry, Mary B.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Steele, Linda; Neuhausen, Susan L.; Ding, Yuan Chun; Hansen, Thomas v. O.; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N.; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K.; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M.; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A.; van Os, Theo A. M.; van der Kolk, Lizet; de Lange, J. L.; Meijers-Heijboer, Hanne E. J.; van der Hout, A. H.; van Asperen, Christi J.; Gómez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collée, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; HEBON; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th.; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R.; Healey, Sue; Investigators, kConFab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A.; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F.; Offit, Kenneth; Couch, Fergus J.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Benitez, Javier

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10−3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10−3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied. PMID:24698998

  11. Dynamic control of strand excision during human DNA mismatch repair.

    PubMed

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-03-22

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

  12. Biomolecular Simulation of Base Excision Repair and Protein Signaling

    SciTech Connect

    Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W

    2006-03-03

    The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.

  13. Free energy profiles of base flipping in intercalative polycyclic aromatic hydrocarbon-damaged DNA duplexes: Energetic and structural relationships to nucleotide excision repair susceptibility

    PubMed Central

    Cai, Yuqin; Zheng, Han; Ding, Shuang; Kropachev, Konstantin; Schwaid, Adam G.; Tang, Yijin; Mu, Hong; Wang, Shenglong; Geacintov, Nicholas E.; Zhang, Yingkai; Broyde, Suse

    2013-01-01

    The crystal structure of Rad4/Rad23, the yeast homolog of the human nucleotide excision repair (NER) lesion recognition factor XPC-RAD23B (Min and Pavletich, (2007) Nature 449:570–575) reveals that the lesion-partner base is flipped out of the helix and binds the protein. This suggests the hypothesis that flipping of this partner base must overcome a free energy barrier, which constitutes one element contributing to changes in the thermodynamic properties induced by the DNA damage and sensed by the recognition protein. We explored this hypothesis by computing complete flipping free energy profiles for two lesions derived from the procarcinogenic polycyclic aromatic hydrocarbons (PAHs), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), R-trans-anti-DB[a,l]P-N6-dA (R-DB[a,l]P-dA) and R-trans-anti-B[a]P-N6-dA (R-B[a]P-dA), and the corresponding unmodified duplex. The DB[a,l]P and B[a]P adducts differ in number and organization of their aromatic rings. We integrate these results with prior profiles for the R-trans-anti-DB[a,l]P-dG adduct (Zheng et al., (2010) Chem. Res. Toxicol. 23:1868–1870). All adopt conformational themes involving intercalation of the PAH aromatic ring system into the DNA duplex; however, R-DB[a,l]P-dA and R-B[a]P-dA intercalate from the major groove, while R-DB[a,l]P-dG intercalates from the minor groove. These structural differences produce different computed van der Waals stacking interaction energies between the flipping partner base with the lesion aromatic ring system and adjacent bases; we find that the better the stacking, the higher the relative flipping free energy barrier and hence lower flipping probability. The better relative NER susceptibilities correlate with greater ease of flipping in these three differently intercalated lesions. In addition to partner base-flipping, the Rad4/Rad23 crystal structure shows that a protein-β-hairpin, BHD3, intrudes from the major groove side between the DNA strands at the lesion site. We

  14. Rational Inhibitors of DNA Base Excision Repair Enzymes: New Tools for Elucidating the Role of BER in Cancer Chemotherapy. Addendum

    DTIC Science & Technology

    2006-11-01

    30. Drohat, A. C., Jagadeesh, J., Ferguson, E., and Stivers, J. T. (1999) The role of electrophilic and base catalysis in the mechanism of Escherichia...based on a duplex previously used in rapid kinetic studies of base flipping by UDG (Figure 2) (5). The 2′ fluorinated deoxyuridine substrate analogue...Boca Raton, FL. 32. Drohat, A. C., Jagadeesh, J., Ferguson, E., and Stivers, J. T. (1999) Role of electrophilic and general base catalysis in the

  15. Repair of oxidatively induced DNA damage by DNA glycosylases: Mechanisms of action, substrate specificities and excision kinetics.

    PubMed

    Dizdaroglu, Miral; Coskun, Erdem; Jaruga, Pawel

    Endogenous and exogenous reactive species cause oxidatively induced DNA damage in living organisms by a variety of mechanisms. As a result, a plethora of mutagenic and/or cytotoxic products are formed in cellular DNA. This type of DNA damage is repaired by base excision repair, although nucleotide excision repair also plays a limited role. DNA glycosylases remove modified DNA bases from DNA by hydrolyzing the glycosidic bond leaving behind an apurinic/apyrimidinic (AP) site. Some of them also possess an accompanying AP-lyase activity that cleaves the sugar-phosphate chain of DNA. Since the first discovery of a DNA glycosylase, many studies have elucidated the mechanisms of action, substrate specificities and excision kinetics of these enzymes present in all living organisms. For this purpose, most studies used single- or double-stranded oligodeoxynucleotides with a single DNA lesion embedded at a defined position. High-molecular weight DNA with multiple base lesions has been used in other studies with the advantage of the simultaneous investigation of many DNA base lesions as substrates. Differences between the substrate specificities and excision kinetics of DNA glycosylases have been found when these two different substrates were used. Some DNA glycosylases possess varying substrate specificities for either purine-derived lesions or pyrimidine-derived lesions, whereas others exhibit cross-activity for both types of lesions. Laboratory animals with knockouts of the genes of DNA glycosylases have also been used to provide unequivocal evidence for the substrates, which had previously been found in in vitro studies, to be the actual substrates in vivo as well. On the basis of the knowledge gained from the past studies, efforts are being made to discover small molecule inhibitors of DNA glycosylases that may be used as potential drugs in cancer therapy.

  16. Autophagy positively regulates DNA damage recognition by nucleotide excision repair.

    PubMed

    Qiang, Lei; Zhao, Baozhong; Shah, Palak; Sample, Ashley; Yang, Seungwon; He, Yu-Ying

    2016-01-01

    Macroautophagy (hereafter autophagy) is a cellular catabolic process that is essential for maintaining tissue homeostasis and regulating various normal and pathologic processes in human diseases including cancer. One cancer-driving process is accumulation of genetic mutations due to impaired DNA damage repair, including nucleotide excision repair. Here we show that autophagy positively regulates nucleotide excision repair through enhancing DNA damage recognition by the DNA damage sensor proteins XPC and DDB2 via 2 pathways. First, autophagy deficiency downregulates the transcription of XPC through TWIST1-dependent activation of the transcription repressor complex E2F4-RBL2. Second, autophagy deficiency impairs the recruitment of DDB2 to ultraviolet radiation (UV)-induced DNA damage sites through TWIST1-mediated inhibition of EP300. In mice, the pharmacological autophagy inhibitor Spautin-1 promotes UVB-induced tumorigenesis, whereas the autophagy inducer rapamycin reduces UVB-induced tumorigenesis. These findings demonstrate the crucial role of autophagy in maintaining proper nucleotide excision repair in mammalian cells and suggest a previously unrecognized tumor-suppressive mechanism of autophagy in cancer.

  17. Lack of association between polymorphisms of the DNA base excision repair genes MUTYH and hOGG1 and keratoconus in a Polish subpopulation

    PubMed Central

    Synowiec, Ewelina; Wójcik, Katarzyna A.; Czubatka, Anna; Polakowski, Piotr; Izdebska, Justyna; Szaflik, Jerzy; Błasiak, Janusz

    2015-01-01

    Introduction Keratoconus (KC) is a non-inflammatory thinning of the cornea and a leading indication for corneal transplantation. Oxidative stress plays a role in the pathogenesis of this disease. The products of the hOGG1 and MUTYH genes play an important role in the repair of oxidatively modified DNA in the base excision repair pathway. We hypothesized that variability in these genes may change susceptibility to oxidative stress and predispose individuals to the development of KC. We investigated the possible association between the c.977C>G polymorphism of the hOGG1 gene (rs1052133) and the c.972G>C polymorphism of the MUTYH gene (rs3219489) and KC occurrence as well as the modulation of this association by some KC risk factors. Material and methods A total of 205 patients with KC and 220 controls were included in this study. The polymorphisms were genotyped with polymerase chain reaction (PCR) restriction fragment length polymorphism and PCR-confronting two-pair primer techniques. Differences in genotype and allele frequency distributions were evaluated using the χ2 test, and KC risk was estimated with an unconditional multiple logistic regression with and without adjustment for co-occurrence of visual impairment, allergies, sex and family history for KC. Results We did not find any association between the genotypes and combined genotypes of the c.977C>G polymorphism of the hOGG1 gene and the c.972G>C polymorphism of the MUTYH gene and the occurrence of KC. Conclusions Our findings suggest that the c.977C>G-hOGG1 polymorphism and the c.972G>C-MUTYH polymorphism may not be linked with KC occurrence in this Polish subpopulation. PMID:26528356

  18. DNA Base Excision Repair (BER) and Cancer Gene Therapy: Use of the Human N-mythlpurien DNA Glycosylase (MPG) to Sensitize Breast Cancer Cells to Low Dose Chemotherapy

    DTIC Science & Technology

    2003-06-01

    has found that the overexpression of this DNA repair protein is cytotoxic to tumor cells in response to the classic alkylating agent, methyl...SUBJECT TERMS 15. NUMBER OF PAGES DNA repair, methylpurine DNA glycosylase, breast cancer 5 16. PRICE CODE 17. SECURITY CLASSIFICATION 18 . SECURITY...NSN 7540-01-280-5500 Standard Form 298 (Rev. 2-89) Prescribed by ANSI Std. Z39- 18 298-102 BEST AVAILABLE COPY Table of Contents Cover

  19. 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.

    PubMed

    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.

  20. The human oxidative DNA glycosylase NEIL1 excises psoralen-induced interstrand DNA cross-links in a three-stranded DNA structure.

    PubMed

    Couvé, Sophie; Macé-Aimé, Gaëtane; Rosselli, Filippo; Saparbaev, Murat K

    2009-05-01

    Previously, we have demonstrated that human oxidative DNA glycosylase NEIL1 excises photoactivated psoralen-induced monoadducts but not genuine interstrand cross-links (ICLs) in duplex DNA. It has been postulated that the repair of ICLs in mammalian cells is mainly linked to DNA replication and proceeds via dual incisions in one DNA strand that bracket the cross-linked site. This process, known as "unhooking," enables strand separation and translesion DNA synthesis through the gap, yielding a three-stranded DNA repair intermediate composed of a short unhooked oligomer covalently bound to the duplex. At present, the detailed molecular mechanism of ICL repair in mammalian cells remains unclear. Here, we constructed and characterized three-stranded DNA structures containing a single ICL as substrates for the base excision repair proteins. We show that NEIL1 excises with high efficiency the unhooked ICL fragment within a three-stranded DNA structure. Complete reconstitution of the repair of unhooked ICL shows that it can be processed in a short patch base excision repair pathway. The new substrate specificity of NEIL1 points to a preferential involvement in the replication-associated repair of ICLs. Based on these data, we propose a model for the mechanism of ICL repair in mammalian cells that implicates the DNA glycosylase activity of NEIL1 downstream of Xeroderma Pigmentosum group F/Excision Repair Cross-Complementing 1 endonuclease complex (XPF/ERCC1) and translesion DNA synthesis repair steps. Finally, our data demonstrate that Nei-like proteins from Escherichia coli to human cells can excise bulky unhooked psoralen-induced ICLs via hydrolysis of glycosidic bond between cross-linked base and deoxyribose sugar, thus providing an alternative heuristic solution for the removal of complex DNA lesions.

  1. Profiling base excision repair glycosylases with synthesized transition state analogs.

    PubMed

    Chu, Aurea M; Fettinger, James C; David, Sheila S

    2011-09-01

    Two base excision repair glycosylase (BER) transition state (TS) mimics, (3R,4R)-1-benzyl (hydroxymethyl) pyrrolidin-3-ol (1NBn) and (3R,4R)-(hydroxymethyl) pyrrolidin-3-ol (1N), were synthesized using an improved method. Several BER glycosylases that repair oxidized DNA bases, bacterial formamidopyrimdine glycosylase (Fpg), human OG glycosylase (hOGG1) and human Nei-like glycosylase 1 (hNEIL1) exhibit exceptionally high affinity (K(d)∼pM) with DNA duplexes containing the 1NBn and 1N nucleotide. Notably, comparison of the K(d) values of both TS mimics relative to an abasic analog (THF) in duplex contexts paired opposite C or A suggest that these DNA repair enzymes use distinctly different mechanisms for damaged base recognition and catalysis despite having overlapping substrate specificities.

  2. Excision of 5-hydroxymethylcytosine by DEMETER family DNA glycosylases

    PubMed Central

    Jang, Hosung; Shin, Hosub; Eichman, Brandt F.; Huh, Jin Hoe

    2016-01-01

    In plants and animals, 5-methylcytosine (5mC) serves as an epigenetic mark to repress gene expression, playing critical roles for cellular differentiation and transposon silencing. Mammals also have 5-hydroxymethylcytosine (5hmC), resulting from hydroxylation of 5mC by TET family-enzymes. 5hmC is abundant in mouse Purkinje neurons and embryonic stem cells, and regarded as an important intermediate for active DNA demethylation in mammals. However, the presence of 5hmC in plants has not been clearly demonstrated. In Arabidopsis, the DEMETER (DME) family DNA glycosylases efficiently remove 5mC, which results in DNA demethylation and transcriptional activation of target genes. Here we show that DME and ROS1 have a significant 5hmC excision activity in vitro, although we detected no 5hmC in Arabidopsis, suggesting that it is very unlikely for plants to utilize 5hmC as a DNA demethylation intermediate. Our results indicate that both plants and animals have 5mC in common but DNA demethylation systems have independently evolved with distinct mechanisms. PMID:24661881

  3. Selective excision of 5-carboxylcytosine by a thymine DNA glycosylase mutant

    PubMed Central

    Hashimoto, Hideharu; Zhang, Xing; Cheng, Xiaodong

    2013-01-01

    The mammalian thymine DNA glycosylase (TDG) excises the mismatched base, uracil, thymine, or 5-hydroxymethyluracil (5hmU), as well as removes 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) when paired with a guanine. In the previously solved structure of TDG in complex with DNA containing 5caC, the side chain of asparagine 157 (N157) contacts the 5-carboxyl moiety of 5caC via a weak hydrogen bond. We examined the role of N157 in recognition of 5caC by mutagenesis. The asparagine-to-alanine (N157A) mutant has no detectable base excision activity for a G:T mismatch, and its excision activity is reduced for other substrates including G:5caC. Unexpectedly, the asparagine-to-aspartate (N157D) mutant has a comparable base excision rate for G:5caC substrate to that of wild type, but it only has residual activity for G:U and no detectable activity for other substrates. We further show that the N157D mutant has higher activity for 5caC at a lower pH (6.0), suggesting that increased protonation of the carboxylate of 5caC and the aspartate facilitates base excision. The N157D mutant remains highly specific for 5caC even in the presence of large excess of genomic DNA, a property that can potentially be used for mapping the very low amount of 5caC in genomes. PMID:23337108

  4. A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine

    PubMed Central

    Ponferrada-Marín, María Isabel; Parrilla-Doblas, Jara Teresa; Roldán-Arjona, Teresa; Ariza, Rafael R.

    2011-01-01

    DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases. PMID:21036872

  5. Rational Inhibitors of DNA Base Excision Repair (BER) Enzymes: New Tools for Elucidating the Role of the BER in Cancer Chemotherapy

    DTIC Science & Technology

    2005-05-01

    right of the bars. (B) Incremental change in binding free energy as UDG (Figure 2) (5). The 2’ fluorinated deoxyuridine each hydrogen bond is removed from...1996) Naphthalene, phenanthrene, and pyrene as DNA h187q Escherichia coli uracil DNA glycosylase: Electrophilic base analogues: Synthesis, structure...solution, Biochemistry 32. Drohat, A. C., Jagadeesh, J., Ferguson, E., and Stivers, J. T. (1999) 26, 5646-5650. Role of electrophilic and general base

  6. Base excision repair in chromatin: Insights from reconstituted systems

    PubMed Central

    Balliano, Angela J.; Hayes, Jeffrey J.

    2015-01-01

    The process of base excision repair has been completely reconstituted in vitro and structural and biochemical properties of the component enzymes thoroughly studied on naked DNA templates. More recent work in this field aims to understand how BER operates on the natural substrate, chromatin [1, 2]. Toward this end, a number of researchers, including the Smerdon group, have focused attention to understand how individual enzymes and reconstituted BER operate on nucleosome substrates. While nucleosomes were once thought to completely restrict access of DNA-dependent factors, the surprising finding from these studies suggests that at least some BER components can utilize target DNA bound within nucleosomes as substrates for their enzymatic processes. This data correlates well with both structural studies of these enzymes and our developing understanding of nucleosome conformation and dynamics. While more needs to be learned, these studies highlight the utility of reconstituted BER and chromatin systems to inform our understanding of in vivo biological processes. PMID:26411876

  7. AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis

    PubMed Central

    Lee, Jiyoon; Jang, Hosung; Shin, Hosub; Choi, Woo Lee; Mok, Young Geun; Huh, Jin Hoe

    2014-01-01

    DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3′-phosphor-α, β-unsaturated aldehyde and 3′-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3′-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants. PMID:25228464

  8. Nucleosomes determine their own patch size in base excision repair

    PubMed Central

    Meas, Rithy; Smerdon, Michael J.

    2016-01-01

    Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2–12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in ‘designed’ nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase β and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER. PMID:27265863

  9. Genetic variants involved in oxidative stress, base excision repair, DNA methylation, and folate metabolism pathways influence myeloid neoplasias susceptibility and prognosis.

    PubMed

    Gonçalves, Ana Cristina; Alves, Raquel; Baldeiras, Inês; Cortesão, Emília; Carda, José Pedro; Branco, Claudia C; Oliveiros, Bárbara; Loureiro, Luísa; Pereira, Amélia; Nascimento Costa, José Manuel; Sarmento-Ribeiro, Ana Bela; Mota-Vieira, Luisa

    2017-01-01

    Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) share common features: elevated oxidative stress, DNA repair deficiency, and aberrant DNA methylation. We performed a hospital-based case-control study to evaluate the association in variants of genes involved in oxidative stress, folate metabolism, DNA repair, and DNA methylation with susceptibility and prognosis of these malignancies. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped in 191 patients (101 MDS and 90 AML) and 261 controls. We also measured oxidative stress (reactive oxygen species/total antioxidant status ratio), DNA damage (8-hydroxy-2'-deoxyguanosine), and DNA methylation (5-methylcytosine) in 50 subjects (40 MDS and 10 controls). Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. We observed a correlation of CYBA TT, GPX1 TT, and SOD2 CC genotypes with increased oxidative stress levels, as well as NEIL1 TT and OGG1 GG genotypes with higher DNA damage. The 5-methylcytosine levels were negatively associated with DNMT1 CC, DNMT3A CC, and MTRR AA genotypes, and positively with DNMT3B CC genotype. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced survival of MDS and AML patients. Altogether, data suggest that genetic variability influence predisposition and prognosis of MDS and AML patients, as well AML transformation rate in MDS patients. © 2016 Wiley Periodicals, Inc.

  10. POLYMORPHISMS IN THE DNA BASE EXCISION REPAIR GENES APEX1 AND XRCC1 AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in revers...

  11. Alar base reduction: the boomerang-shaped excision.

    PubMed

    Foda, Hossam M T

    2011-04-01

    A boomerang-shaped alar base excision is described to narrow the nasal base and correct the excessive alar flare. The boomerang excision combined the external alar wedge resection with an internal vestibular floor excision. The internal excision was inclined 30 to 45 degrees laterally to form the inner limb of the boomerang. The study included 46 patients presenting with wide nasal base and excessive alar flaring. All cases were followed for a mean period of 18 months (range, 8 to 36 months). The laterally oriented vestibular floor excision allowed for maximum preservation of the natural curvature of the alar rim where it meets the nostril floor and upon its closure resulted in a considerable medialization of alar lobule, which significantly reduced the amount of alar flare and the amount of external alar excision needed. This external alar excision measured, on average, 3.8 mm (range, 2 to 8 mm), which is significantly less than that needed when a standard vertical internal excision was used ( P < 0.0001). Such conservative external excisions eliminated the risk of obliterating the natural alar-facial crease, which did not occur in any of our cases. No cases of postoperative bleeding, infection, or vestibular stenosis were encountered. Keloid or hypertrophic scar formation was not encountered; however, dermabrasion of the scars was needed in three (6.5%) cases to eliminate apparent suture track marks. The boomerang alar base excision proved to be a safe and effective technique for narrowing the nasal base and elimination of the excessive flaring and resulted in a natural, well-proportioned nasal base with no obvious scarring.

  12. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

    PubMed

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Jia, Lei; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue-DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision

  13. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

    PubMed Central

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide

  14. DNA base-excision repair enzyme apurinic/apyrimidinic endonuclease/redox factor-1 is increased and competent in the brain and spinal cord of individuals with amyotrophic lateral sclerosis.

    PubMed

    Shaikh, Arif Y; Martin, Lee J

    2002-01-01

    Motor neurons degenerate in amyotrophic lateral sclerosis (ALS). The mechanisms for this neuronal cell death are not known, although apoptosis has been implicated. Oxidative damage to DNA and activation of p53 has been identified directly in motor neurons in cases of ALS. We evaluated whether motor neuron degeneration in ALS is associated with changes in the levels and function of the multifunctional protein apurinic/apyrimidinic endonuclease (APE/Ref-1). APE/Ref-1 functions as an enzyme in the DNA base-excision repair pathway and as a redox-regulation protein for transcription factors. The protein level and localization of APE/Ref-1 are changed in ALS. Immunoblotting showed that APE/Ref-1 protein levels are increased in selectively vulnerable central nervous system (CNS) regions in individuals with ALS compared to age-matched controls. Plasmid DNA repair assay demonstrated that APE from individuals with ALS is competent in repairing apurinic (AP) sites. DNA repair function in nuclear fractions is increased significantly in ALS motor cortex and spinal cord. Immunocytochemistry and single-cell densitometry revealed that APE/Ref-1 is expressed at lower levels in control motor neurons than in ALS motor neurons, which are decreased in number by 42% in motor cortex. APE/Ref-1 is increased in the nucleus of remaining upper motor neurons in ALS, which show a 38% loss of nuclear area. APE-Ref-1 is also upregulated in astrocytes in spinal cord white matter pathways in familial ALS. We conclude that mechanisms for DNA repair are activated in ALS, supporting the possibility that DNA damage is an upstream mechanism for motor neuron degeneration in this disease.

  15. An unprecedented nucleic acid capture mechanism for excision of DNA damage

    SciTech Connect

    Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.; Gold, Barry; Eichmanbrand, Brandt F.

    2010-11-18

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

  16. Base excision repair: A critical player in many games

    PubMed Central

    Wallace, Susan S.

    2014-01-01

    This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person’s view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field. PMID:24780558

  17. Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro.

    PubMed Central

    Sancar, A; Franklin, K A; Sancar, G B

    1984-01-01

    Pyrimidine dimers are the major photoproducts produced in cellular DNA upon UV irradiation. In Escherichia coli there are dark and photorepair mechanisms that eliminate the dimers from DNA and prevent their lethal and mutagenic effects. To determine whether these repair mechanisms act cooperatively or competitively in repairing DNA, we investigated the effects upon one another of DNA photolyase, which mediates photorepair, and uvrABC excision nuclease, an enzyme complex of the uvrABC gene products, which catalyzes nucleotide excision repair. We found that photolyase stimulates the removal of pyrimidine dimers but not other DNA adducts by uvrABC excision nuclease. The two subunits of uvrABC excision nuclease, the uvrA and uvrB proteins which together bind to the dimer region of DNA, had no effect on the activity of photolyase. T4 endonuclease V, which like photolyase is specific for pyrimidine dimers, was inhibited by photolyase, suggesting that these two proteins recognize the same or similar chemical structures in UV-irradiated DNA that are different from those recognized by uvrABC excision nuclease. Images PMID:6390436

  18. Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging.

    PubMed

    Swain, Umakanta; Subba Rao, Kalluri

    2011-08-01

    Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as "comet" assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies.

  19. Base sequence context effects on nucleotide excision repair.

    PubMed

    Cai, Yuqin; Patel, Dinshaw J; Broyde, Suse; Geacintov, Nicholas E

    2010-08-23

    Nucleotide excision repair (NER) plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P), 10S (+)-trans-anti-B[a]P-N(2)-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair.

  20. Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence.

    PubMed

    Allgayer, Julia; Kitsera, Nataliya; von der Lippen, Carina; Epe, Bernd; Khobta, Andriy

    2013-10-01

    8-Oxoguanine (8-oxoG) is a major product of oxidative DNA damage, which induces replication errors and interferes with transcription. By varying the position of single 8-oxoG in a functional gene and manipulating the nucleotide sequence surrounding the lesion, we found that the degree of transcriptional inhibition is independent of the distance from the transcription start or the localization within the transcribed or the non-transcribed DNA strand. However, it is strongly dependent on the sequence context and also proportional to cellular expression of 8-oxoguanine DNA glycosylase (OGG1)-demonstrating that transcriptional arrest does not take place at unrepaired 8-oxoG and proving a causal connection between 8-oxoG excision and the inhibition of transcription. We identified the 5'-CAGGGC[8-oxoG]GACTG-3' motif as having only minimal transcription-inhibitory potential in cells, based on which we predicted that 8-oxoG excision is particularly inefficient in this sequence context. This anticipation was fully confirmed by direct biochemical assays. Furthermore, in DNA containing a bistranded Cp[8-oxoG]/Cp[8-oxoG] clustered lesion, the excision rates differed between the two strands at least by a factor of 9, clearly demonstrating that the excision preference is defined by the DNA strand asymmetry rather than the overall geometry of the double helix or local duplex stability.

  1. Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin.

    PubMed

    Goffin, C; Verly, W G

    1982-10-01

    Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.

  2. High resolution mapping of modified DNA nucleobases using excision repair enzymes

    PubMed Central

    Bryan, D. Suzi; Ransom, Monica; Adane, Biniam; York, Kerri

    2014-01-01

    The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes. PMID:25015380

  3. Gut Microbiota Imbalance and Base Excision Repair Dynamics in Colon Cancer

    PubMed Central

    Ray, Debolina; Kidane, Dawit

    2016-01-01

    Gut microbiota are required for host nutrition, energy balance, and regulating immune homeostasis, however, in some cases, this mutually beneficial relationship becomes twisted (dysbiosis), and the gut flora can incite pathological disorders including colon cancer. Microbial dysbiosis promotes the release of bacterial genotoxins, metabolites, and causes chronic inflammation, which promote oxidative DNA damage. Oxidized DNA base lesions are removed by base excision repair (BER), however, the role of this altered function of BER, as well as microbiota-mediated genomic instability and colon cancer development, is still poorly understood. In this review article, we will discuss how dysbiotic microbiota induce DNA damage, its impact on base excision repair capacity, the potential link of host BER gene polymorphism, and the risk of dysbiotic microbiota mediated genomic instability and colon cancer. PMID:27471558

  4. Roles of Exc protein and DNA homology in the CTnDOT excision reaction.

    PubMed

    Keeton, Carolyn M; Gardner, Jeffrey F

    2012-07-01

    Excision from the chromosome is the first step during the transfer of conjugative transposons (CTns) to a recipient. We previously showed that the excision of CTnDOT is more complex than the excision of lambdoid phages and CTns such as Tn916. The excision in vivo of CTnDOT utilizes four CTnDOT-encoded proteins, IntDOT, Xis2c, Xis2d, and Exc, and a host factor. We previously developed an in vitro excision reaction where the recombination sites attL and attR were located on different plasmids. The reaction was inefficient and did not require Exc, suggesting that the reaction conditions did not mimic in vivo conditions. Here, we report the development of an intramolecular excision reaction where the attL and attR sites are located on the same DNA molecule. We found that Exc stimulates the reaction 3- to 5-fold. The efficiency of the excision reaction was also dependent on the distance between the attL and attR sites and on the sequences of the overlap regions between the sites of the strand exchanges. Substrates with identical overlap sequences recombined more efficiently than ones with heterologous overlap sequences. This was surprising, because the integration reaction is not sensitive to heterology in the overlap regions of the attDOT and attB sites.

  5. Regulation of base excision repair in eukaryotes by dynamic localization strategies.

    PubMed

    Swartzlander, Daniel B; Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

    2012-01-01

    This chapter discusses base excision repair (BER) and the known mechanisms defined thus far regulating BER in eukaryotes. Unlike the situation with nucleotide excision repair and double-strand break repair, little is known about how BER is regulated to allow for efficient and accurate repair of many types of DNA base damage in both nuclear and mitochondrial genomes. Regulation of BER has been proposed to occur at multiple, different levels including transcription, posttranslational modification, protein-protein interactions, and protein localization; however, none of these regulatory mechanisms characterized thus far affect a large spectrum of BER proteins. This chapter discusses a recently discovered mode of BER regulation defined in budding yeast cells that involves mobilization of DNA repair proteins to DNA-containing organelles in response to genotoxic stress.

  6. Precise excision of plastid DNA by the large serine recombinase Bxb1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To elucidate the precise excision of transgene, tobacco plastid genome was transformed with a vector (pTCH-BxbPB) that contains a stuffer DNA fragment flanked by directly oriented attB and attP recognition sites for the Bxb1 recombinase. The transformed plastid genomes containing the recognition si...

  7. Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles.

    PubMed

    Weng, Liwei; Greenberg, Marc M

    2015-09-02

    C5'-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5'-radical. DOB is a potent irreversible inhibitor of DNA polymerase β, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275-1500-fold faster than that in free DNA. The half-life of DOB (7.0-16.8 min) in NCPs is shorter than any other abasic lesion. DOB's lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.

  8. FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC

    PubMed Central

    Ouararhni, Khalid; Roulland, Yohan; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan

    2016-01-01

    FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER. PMID:27467129

  9. Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends.

    PubMed

    Zhao, Boyang; O'Brien, Patrick J

    2011-05-24

    The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.

  10. Role of ATP in UV-induced DNA excision repair in human cells

    SciTech Connect

    Dresler, S.L.

    1986-05-01

    In permeable human fibroblasts, UV-induced DNA excision repair is dependent on ATP, with a K/sub m/ of approximately 1 mM. Omission of ATP from the reaction mix completely inhibits damage-specific incision of DNA, but has little effect on repair patch synthesis proceeding from previously incised sites. UV-induced excision repair in permeable xeroderma pigmentosum (XP) cells complemented with T4 UV endonuclease is also totally dependent on ATP. Because the T4 enzyme is not ATP-dependent, ATP must be required for an endogenous activity other than the incision of damaged DNA. Alkaline elution reveals that, in the absence of ATP, T4 UV endonuclease does incise the DNA of permeable UV-irradiated XP cells, but that the incision rate is stimulated approximately 2-fold by the addition of ATP. This 2-fold stimulation of incision can not, however, be responsible for the absolute ATP dependence of excision repair in UV endonuclease-complemented XP cells. Apparently, although T4 UV endonuclease can incise damaged nuclear DNA in the absence of ATP, the incised sites must also be altered in an ATP-dependent reaction before subsequent steps of the repair process can proceed. This conclusion, coupled with the fact that ATP stimulates incision of damaged nuclear DNA by T4 UV endonuclease and is absolutely required for incision of damaged nuclear DNA by the endogenous human UV endonuclease, suggests that an important function of the early ATP-dependent step in UV-induced excision repair is to make damaged sites in DNA accessible to repair enzymes.

  11. Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

    PubMed Central

    Çağlayan, Melike; Horton, Julie K.; Dai, Da-Peng; Stefanick, Donna F.; Wilson, Samuel H.

    2017-01-01

    Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides. PMID:28067232

  12. Base Excision Repair in Physiology and Pathology of the Central Nervous System

    PubMed Central

    Bosshard, Matthias; Markkanen, Enni; van Loon, Barbara

    2012-01-01

    Relatively low levels of antioxidant enzymes and high oxygen metabolism result in formation of numerous oxidized DNA lesions in the tissues of the central nervous system. Accumulation of damage in the DNA, due to continuous genotoxic stress, has been linked to both aging and the development of various neurodegenerative disorders. Different DNA repair pathways have evolved to successfully act on damaged DNA and prevent genomic instability. The predominant and essential DNA repair pathway for the removal of small DNA base lesions is base excision repair (BER). In this review we will discuss the current knowledge on the involvement of BER proteins in the maintenance of genetic stability in different brain regions and how changes in the levels of these proteins contribute to aging and the onset of neurodegenerative disorders. PMID:23203191

  13. Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions

    PubMed Central

    Tsaalbi-Shtylik, Anastasia; Ferrás, Cristina; Pauw, Bea; Hendriks, Giel; Temviriyanukul, Piya; Carlée, Leone; Calléja, Fabienne; van Hees, Sandrine; Akagi, Jun-Ichi; Iwai, Shigenori; Hanaoka, Fumio; Jansen, Jacob G.

    2015-01-01

    In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa–Atr–Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects. PMID:25869665

  14. Crystal structure of the DNA nucleotide excision repair enzyme UvrB from Thermus thermophilus

    PubMed Central

    Machius, Mischa; Henry, Lisa; Palnitkar, Maya; Deisenhofer, Johann

    1999-01-01

    Nucleotide excision repair (NER) is the most important DNA-repair mechanism in living organisms. In prokaryotes, three enzymes forming the UvrABC system initiate NER of a variety of structurally different DNA lesions. UvrB, the central component of this system, is responsible for the ultimate DNA damage recognition and participates in the incision of the damaged DNA strand. The crystal structure of Thermus thermophilus UvrB reveals a core that is structurally similar to core regions found in helicases, where they constitute molecular motors. Additional domains implicated in binding to DNA and various components of the NER system are attached to this central core. The architecture and distribution of DNA binding sites suggest a possible model for the DNA damage recognition process. PMID:10518516

  15. Studying nucleotide excision repair of mammalian DNA in a cell-free system

    SciTech Connect

    Wood, R.D.

    1994-12-31

    During nucleotide excision repair, a multiprotein system locates a lesion in DNA and catalyzes enzymatic cleavage of the altered strand. The damaged oligonucleotide and the incision proteins are then displaced, DNA synthesis proceeds to form a short patch using the nonmodified strand as a template, and repair is completed by a DNA ligase. Many gene products participate in these reactions, the best known of which correspond to the seven genetic complementation groups XP-A to XP-G of the disease xeroderma pigmentosum (XP). Cells representing any of these XP groups appear to exhibit, to varying degrees, defects in the first steps of nucleotide excision repair. Individuals affected with XP are hypersensitive to sunlight; most have a predisposition to skin cancer, and some patients show severe neurological abnormalities. In addition to XP, other UV-sensitive mutants of mammalian cells are providing insight into nucleotide excision repair. Of particular interest are mutants isolated from the rodent cells, which have been assigned to 11 different complementation groups. Human genes that can correct the repair defects of rodent mutants in these complementation groups are denoted. ERCC (excision repair cross-complementing) genes are are referred to by number, ERCC1 to ERCC11. Some of these genes are proving to be equivalent to particular XP-complementing genes, while others are distinct. The process of nucleotide excision repair is evolutionarily conserved in eukaryotes, and functional homologues of many of the ERCC and XP genes have been identified in other organisms; studies in yeast are proving to be particularly informative.

  16. Functions of disordered regions in mammalian early base excision repair proteins

    PubMed Central

    Hegde, Muralidhar L.; Hazra, Tapas K.

    2010-01-01

    Reactive oxygen species, generated endogenously and induced as a toxic response, produce several dozen oxidized or modified bases and/or single-strand breaks in mammalian and other genomes. These lesions are predominantly repaired via the conserved base excision repair (BER) pathway. BER is initiated with excision of oxidized or modified bases by DNA glycosylases leading to formation of abasic (AP) site or strand break at the lesion site. Structural analysis by experimental and modeling approaches shows the presence of a disordered segment commonly localized at the N- or C-terminus as a characteristic signature of mammalian DNA glycosylases which is absent in their bacterial prototypes. Recent studies on unstructured regions in DNA metabolizing proteins have indicated their essential role in interaction with other proteins and target DNA recognition. In this review, we have discussed the unique presence of disordered segments in human DNA glycosylases, and AP endonuclease involved in the processing of glycosylase products, and their critical role in regulating repair functions. These disordered segments also include sites for posttranslational modifications and nuclear localization signal. The teleological basis for their structural flexibility is discussed. PMID:20714778

  17. Early days of DNA repair: discovery of nucleotide excision repair and homology-dependent recombinational repair.

    PubMed

    Rupp, W Dean

    2013-12-13

    The discovery of nucleotide excision repair in 1964 showed that DNA could be repaired by a mechanism that removed the damaged section of a strand and replaced it accurately by using the remaining intact strand as the template. This result showed that DNA could be actively metabolized in a process that had no precedent. In 1968, experiments describing postreplication repair, a process dependent on homologous recombination, were reported. The authors of these papers were either at Yale University or had prior Yale connections. Here we recount some of the events leading to these discoveries and consider the impact on further research at Yale and elsewhere.

  18. Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study.

    PubMed

    Kaminski, R; Bella, R; Yin, C; Otte, J; Ferrante, P; Gendelman, H E; Li, H; Booze, R; Gordon, J; Hu, W; Khalili, K

    2016-08-01

    A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

  19. Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair*

    PubMed Central

    Krasikova, Yuliya S.; Rechkunova, Nadejda I.; Maltseva, Ekaterina A.; Pestryakov, Pavel E.; Petruseva, Irina O.; Sugasawa, Kaoru; Chen, Xuejing; Min, Jung-Hyun; Lavrik, Olga I.

    2013-01-01

    The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (KD ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nm, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms. PMID:23443653

  20. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

    PubMed

    Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

    2015-10-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

  1. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

    PubMed Central

    Fonseca, A.S.; Campos, V.M.A.; Magalhães, L.A.G.; Paoli, F.

    2015-01-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337

  2. Double strand breaks in DNA inhibit nucleotide excision repair in vitro.

    PubMed

    Calsou, P; Frit, P; Salles, B

    1996-11-01

    Nucleotide excision repair (NER) was measured in human cell extracts incubated with either supercoiled or linearized damaged plasmid DNA as repair substrate. NER, as quantified by the extent of repair synthesis activity, was reduced by up to 80% in the case of linearized plasmid DNA when compared with supercoiled DNA. An excess of undamaged linearized plasmid in the repair mixture did not interfere with DNA repair synthesis activity on a supercoiled damaged plasmid, indicating a cis-acting inhibiting effect. In contrast, gaps on circular or linearized plasmids were filled in identically by the DNA polymerases operating in the extracts. When the extent of damage-dependent incision activity was measured, a approximately 70% reduction of repair incision activity by human cell extract was observed on linearized damaged plasmids. Recessed, protruding, or blunt ends were similarly inhibitory. NER activity was partly restored when the extracts were preincubated with autoimmune human sera containing antibodies against the nuclear DNA end-binding heterodimer Ku. In addition, the inhibition of repair activity on linear damaged plasmids was released in extracts from rodent cells deficient in Ku activity but not in extracts from murine scid cells devoid of Ku-associated DNA-dependent kinase activity.

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

    SciTech Connect

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  4. Endoscopic Excision of Symptomatic Simple Bone Cyst at Skull Base

    PubMed Central

    Gunawat, Prashant; Karmarkar, Vikram; Deopujari, Chandrashekhar; Shah, Nishit

    2016-01-01

    Seizure is a classical feature of intra axial brain parenchymal lesion. Simple bone cyst is an unusual bony pathology at skull base presenting with unexpected symptoms of complex partial seizures. Skull base neuro-endoscopy has managed such lesions more effectively with reduced post-operative morbidity as compared to transcranial approach. This case report discusses a 20-year-old male who presented with 3 episodes of seizure over a time period of 10 months. MRI brain revealed T1 hypo and T2 hyper intense cystic lesion in middle cranial fossa with no enhancement on contrast administration. CT scan showed cystic lesion involving greater wing and pterygoid plate of sphenoid on left side. CT cisternographic evaluation showed CSF outpouching in the sphenoid air sinus. Excision of the cystic lesion was carried out through endoscopic transmaxillary transpterygoid approach. Histopathological examination showed the lesion to be a simple bone cyst. PMID:27891396

  5. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    PubMed Central

    Gracheva, Ekaterina; Chitale, Shalaka; Wilhelm, Thomas; Rapp, Alexander; Byrne, Jonathan; Stadler, Jens; Medina, Rebeca; Cardoso, M. Cristina

    2016-01-01

    Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. PMID:27091446

  6. Discovery and Characterization of DNA Excision Repair Pathways: the Work of Philip Courtland Hanawalt

    PubMed Central

    Kresge, Nicole; Simoni, Robert D.; Hill, Robert L.

    2010-01-01

    Expression of Wild-type p53 Is Required for Efficient Global Genomic Nucleotide Excision Repair in UV-irradiated Human Fibroblasts (Ford, J. M., and Hanawalt, P. C. (1997) J. Biol. Chem. 272, 28073–28080) Structural Characterization of RNA Polymerase II Complexes Arrested by a Cyclobutane Pyrimidine Dimer in the Transcribed Strand of Template DNA (Tornaletti, S., Reines, D., and Hanawalt, P. C. (1999) J. Biol. Chem. 274, 24124–24130) G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II (Tornaletti, S., Park-Snyder, S., and Hanawalt, P. C. (2008) J. Biol. Chem. 283, 12756–12762) PMID:20740724

  7. XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells

    SciTech Connect

    Wu Qiangen; Beland, Frederick A.; Chang, Ching-Wei; Fang Jialong

    2011-03-01

    Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 {mu}M AZT treatment significantly increased [{sup 3}H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.

  8. Rev1 is a base excision repair enzyme with 5′-deoxyribose phosphate lyase activity

    PubMed Central

    Prasad, Rajendra; Poltoratsky, Vladimir; Hou, Esther W.; Wilson, Samuel H.

    2016-01-01

    Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5′-deoxyribose phosphate (5′-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro. The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro. The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity. PMID:27683219

  9. Enhanced nucleotide excision repair capacity in lung cancer cells by preconditioning with DNA-damaging agents.

    PubMed

    Choi, Ji Ye; Park, Jeong-Min; Yi, Joo Mi; Leem, Sun-Hee; Kang, Tae-Hong

    2015-09-08

    The capacity of tumor cells for nucleotide excision repair (NER) is a major determinant of the efficacy of and resistance to DNA-damaging chemotherapeutics, such as cisplatin. Here, we demonstrate that using lesion-specific monoclonal antibodies, NER capacity is enhanced in human lung cancer cells after preconditioning with DNA-damaging agents. Preconditioning of cells with a nonlethal dose of UV radiation facilitated the kinetics of subsequent cisplatin repair and vice versa. Dual-incision assay confirmed that the enhanced NER capacity was sustained for 2 days. Checkpoint activation by ATR kinase and expression of NER factors were not altered significantly by the preconditioning, whereas association of XPA, the rate-limiting factor in NER, with chromatin was accelerated. In preconditioned cells, SIRT1 expression was increased, and this resulted in a decrease in acetylated XPA. Inhibition of SIRT1 abrogated the preconditioning-induced predominant XPA binding to DNA lesions. Taking these data together, we conclude that upregulated NER capacity in preconditioned lung cancer cells is caused partly by an increased level of SIRT1, which modulates XPA sensitivity to DNA damage. This study provides some insights into the molecular mechanism of chemoresistance through acquisition of enhanced DNA repair capacity in cancer cells.

  10. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

    PubMed Central

    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

  11. UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro

    SciTech Connect

    Vijg, J.; Mullaart, E.; Berends, F.; Lohman, P.H.; Knook, D.L.

    1986-12-01

    UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.

  12. SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

    PubMed Central

    Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru

    2015-01-01

    The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670

  13. Generation of DNA single-strand displacement by compromised nucleotide excision repair

    PubMed Central

    Godon, Camille; Mourgues, Sophie; Nonnekens, Julie; Mourcet, Amandine; Coin, Fréderic; Vermeulen, Wim; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina

    2012-01-01

    Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients. PMID:22863773

  14. Different structural states in oligonucleosomes are required for early versus late steps of base excision repair

    PubMed Central

    Nakanishi, Shima; Prasad, Rajendra; Wilson, Samuel H.; Smerdon, Michael

    2007-01-01

    Chromatin in eukaryotic cells is folded into higher order structures of folded nucleosome filaments, and DNA damage occurs at all levels of this structural hierarchy. However, little is known about the impact of higher order folding on DNA repair enzymes. We examined the catalytic activities of purified human base excision repair (BER) enzymes on uracil-containing oligonucleosome arrays, which are folded primarily into 30 nm structures when incubated in repair reaction buffers. The catalytic activities of uracil DNA glycosylase (UDG) and apyrimidinic/apurinic endonuclease (APE) digest G:U mismatches to completion in the folded oligonucleosomes without requiring significant disruption. In contrast, DNA polymerase β (Pol β) synthesis is inhibited in a major fraction (∼80%) of the oligonucleosome array, suggesting that single strand nicks in linker DNA are far more accessible to Pol β in highly folded oligonucleosomes. Importantly, this barrier in folded oligonucleosomes is removed by purified chromatin remodeling complexes. Both ISW1 and ISW2 from yeast significantly enhance Pol β accessibility to the refractory nicked sites in oligonucleosomes. These results indicate that the initial steps of BER (UDG and APE) act efficiently on highly folded oligonucleosome arrays, and chromatin remodeling may be required for the latter steps of BER in intact chromatin. PMID:17576692

  15. Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system.

    PubMed

    Long, Dingpei; Lu, Weijian; Hao, Zhanzhang; Xiang, Zhonghuai; Zhao, Aichun

    2016-12-01

    Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.

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

    PubMed

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

    2016-01-08

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

  17. Base excision repair: contribution to tumorigenesis and target in anticancer treatment paradigms

    PubMed Central

    Illuzzi, Jennifer L.; Wilson, David M.

    2015-01-01

    Cancer treatments often lose their effectiveness due to the development of multiple drug resistance. Thus, identification of key proteins involved in the tumorigenic process and the survival mechanism(s), coupled with the design of novel therapeutic compounds (such as small molecule inhibitors), are essential steps towards the establishment of improved anticancer treatment strategies. DNA repair pathways and their proteins have been exposed as potential targets for combinatorial anticancer therapies that involve DNA-interactive cytotoxins, such as alkylating agents, because of their central role in providing resistance against DNA damage. In addition, an understanding of the tumor-specific genetics and associated DNA repair capacity has allowed research scientists and clinicians to begin to devise more targeted treatment strategies based on the concept of synthetic lethality. In this review, the repair mechanisms, as well as the links to cancer progression and treatment, of three key proteins that function in the base excision repair pathway, i.e. APE1, POLβ, and FEN1, are discussed. PMID:22788768

  18. Base excision repair of tandem modifications in a methylated CpG dinucleotide.

    PubMed

    Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S; Beard, William A; Wilson, Samuel H

    2014-05-16

    Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.

  19. Excision without excision

    SciTech Connect

    Brown, David; Sarbach, Olivier; Schnetter, Erik; Diener, Peter; Tiglio, Manuel; Hawke, Ian; Pollney, Denis

    2007-10-15

    to turducken (turduckens, turduckening, turduckened, turduckened) [math.]: To stuff a black hole. We analyze and apply an alternative to black hole excision based on smoothing the interior of black holes with arbitrary initial data, and solving the vacuum Einstein evolution equations everywhere. By deriving the constraint propagation system for our hyperbolic formulation of the BSSN evolution system we rigorously prove that the constraints propagate causally and so any constraint violations introduced inside the black holes cannot affect the exterior spacetime. We present evolutions of Cook-Pfeiffer binary black hole initial configurations showing that these techniques appear to work robustly for generic data. We also present evidence from spherically symmetric evolutions that for the gauge conditions used the same stationary end-state is approached irrespective of the choice of initial data and smoothing procedure.

  20. Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability.

    PubMed

    Markkanen, Enni; Fischer, Roman; Ledentcova, Marina; Kessler, Benedikt M; Dianov, Grigory L

    2015-04-20

    Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells.

  1. Base excision repair activities in organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation.

    PubMed

    Rolseth, Veslemøy; Rundén-Pran, Elise; Neurauter, Christine Gran; Yndestad, Arne; Luna, Luisa; Aukrust, Pål; Ottersen, Ole Petter; Bjørås, Magnar

    2008-06-01

    The capacity for DNA repair is likely to be one of the factors that determine the vulnerability of neurons to ischemic stress and may influence the pathological outcome of stroke. In this report, initiation of base excision repair (BER) was assessed by analysis of enzyme activity and gene expression level of DNA glycosylases and AP-endonucleases in rat organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) - an in vitro model of stroke. Under basal conditions, AP-endonuclease activity and base removal of ethenoadenine and 8-oxoguanine (8-oxoG) were higher (by approximately 20-35 %) in CA3/fascia dentata (FD) than in CA1. Base removal of uracil did not differ between the two hippocampal regions, while removal of 5-hydroxyuracil (5-OHU) was slightly less efficient in CA3/FD than in CA1. Analyses performed immediately after 30 min of OGD revealed a decreased AP-endonuclease activity (by approximately 20%) in CA1 as well as CA3/FD, and an increased ethenoadenine activity (by approximately 25%) in CA1. Activities for 8-oxoG, 5-OHU and uracil showed no significant changes at this time point. At 8h after OGD, none of the enzyme activities differed from control values. Real-time RT-PCR showed that transcription of DNA glycosylases, including Ogg1, Nth1, Ung, Aag, Neil1 and Neil2 were not changed in response to OGD treatment (t=0 h). The hippocampal expression of Neil2 was low compared with the other DNA glycosylases. These data indicate that CA1 has a lower capacity than CA3/FD for removal of base lesions under basal conditions. The relatively low capacity for BER in basal conditions and the apparent failure to upregulate repair of oxidative damage after OGD might contribute to the high vulnerability of CA1 to ischemic injury.

  2. Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA.

    PubMed

    Camenisch, Ulrike; Dip, Ramiro; Vitanescu, Mirela; Naegeli, Hanspeter

    2007-12-01

    The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.

  3. An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS.

    PubMed

    Choi, Jun-Hyuk; Kim, So-Young; Kim, Sook-Kyung; Kemp, Michael G; Sancar, Aziz

    2015-11-27

    DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.

  4. The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase.

    PubMed

    Kuznetsov, N A; Kiryutin, A S; Kuznetsova, A A; Panov, M S; Barsukova, M O; Yurkovskaya, A V; Fedorova, O S

    2017-04-01

    Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.

  5. A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair

    PubMed Central

    Steininger, Sylvia; Ahne, Fred; Winkler, Klaudia; Kleinschmidt, Anja; Eckardt-Schupp, Friederike; Moertl, Simone

    2010-01-01

    The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity. PMID:20040573

  6. Conserved XPB Core Structure and Motifs for DNA Unwinding:Implications for Pathway Selection of Transcription or ExcisionRepair

    SciTech Connect

    Fan, Li; Arval, Andrew S.; Cooper, Priscilla K.; Iwai, Shigenori; Hanaoka, Fumio; Tainer, John A.

    2005-04-01

    The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with Implications for XPB damage verification in nucleotide excision repair.

  7. Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

    PubMed Central

    Krasikova, Yuliya S.; Rechkunova, Nadejda I.; Maltseva, Ekaterina A.; Petruseva, Irina O.; Lavrik, Olga I.

    2010-01-01

    The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG. PMID:20693538

  8. The 8, 5’-Cyclopurine-2’-Deoxynucleosides: Candidate Neurodegenerative DNA Lesions in Xeroderma Pigmentosum, and Unique Probes of Transcription and Nucleotide Excision Repair

    PubMed Central

    Brooks, P. J.

    2008-01-01

    It is a commonly held view that oxidatively-induced DNA lesions are repaired by the base excision repair (BER) pathway, whereas DNA lesions induced by UV light and other “bulky” chemical adducts are repaired by the nucleotide excision repair (NER) pathway. While this distinction is generally accurate, the 8,5’ cyclopurine deoxynucleosides represent an important exception, in that they are formed in DNA by the hydroxyl radical, but are specifically repaired by NER, not by BER. They are also strong blocks to nucleases and polymerases, including RNA polymerase II in human cells. In this review, I will discuss the evidence that these lesions are in part responsible for the neurodegeneration that occurs in some XP patients, and what additional evidence would be necessary to prove such a role. I will also consider other DNA lesions that might be involved in XP neurologic disease. Finally, I will also discuss how our recent studies of these lesions have generated novel insights into the process of transcriptional mutagenesis in human cells, as well as the value of studying these lesions not only for a better understanding of NER, but also for other aspects of human health and disease. PMID:18495558

  9. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies

    PubMed Central

    Shukla, Ankita; Singh, Tiratha Raj

    2016-01-01

    DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC). Since lynch syndrome carries high risk (~40–60%) for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER) and mismatch repair (MMR). Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV) and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels. PMID:27276067

  10. The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway.

    PubMed

    Zhang, Y-M; Liu, J-K; Wong, T-Y

    2003-06-01

    Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.

  11. Excision of ultraviolet damage and the effect of irradiation on DNA synthesis in a strain of Bloom's syndrome fibroblasts

    SciTech Connect

    Henson, P.; Selsky, C.A.; Little, J.B.

    1981-03-01

    Researchers have studied repair of ultraviolet light-induced damage in a strain of Bloom's syndrome cells which we have shown to be defective in host cell reactivation of uv-irradiated herpes simplex virus. Excision repair was monitored by following loss of sensitivity of DNA in permeabilized cells to digestion by the Micrococcus luteus uv endonuclease preparation. The Bloom's syndrome fibroblasts apparently removed endonuclease-sensitive sites from the DNA slightly less efficiently than did normal strains. After 24 h, 38% of the sites remained in the Bloom's syndrome cells in comparison with 16% in normal fibroblasts. DNA newly synthesized in uv-irradiated Bloom's syndrome cells sedimented less far into alkaline sucrose gradients than did DNA from similarly treated normal cells. In other respects, including the effect of caffeine exposure, DNA synthesis in Bloom's syndrome cells was indistinguishable from that in normal cells. We were therefore able to detect only minor defects in the repair of uv-induced damage in Bloom's syndrome fibroblasts. This is consistent with the normal survival exhibited by these cells. The defect in excision repair may, however, be sufficient to allow the cellular repair capacity to become saturated at high infecting multiplicities of uv-irradiated herpes simplex virus.

  12. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    SciTech Connect

    Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi; Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok; Kang, Tae-Hong

    2015-06-05

    Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated in the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.

  13. Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light

    SciTech Connect

    Miller, R.D.; Prakash, L.; Prakash, S.

    1982-08-01

    Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.

  14. Recovery of DNA Replication in UV-Irradiated Escherichia coli Requires both Excision Repair and RecF Protein Function†

    PubMed Central

    Courcelle, Justin; Crowley, David J.; Hanawalt, Philip C.

    1999-01-01

    After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene. In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication. PMID:9922256

  15. Crosstalk between mismatch repair and base excision repair in human gastric cancer.

    PubMed

    Simonelli, Valeria; Leuzzi, Giuseppe; Basile, Giorgia; D'Errico, Mariarosaria; Fortini, Paola; Franchitto, Annapaola; Viti, Valentina; Brown, Ashley R; Parlanti, Eleonora; Pascucci, Barbara; Palli, Domenico; Giuliani, Alessandro; Palombo, Fabio; Sobol, Robert W; Dogliotti, Eugenia

    2016-06-20

    DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase β (Polβ). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Polβ or Polβ active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Polβ were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O6-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Polβ were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Polβ is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Polβ that modulates the response to alkylation damage. These studies suggest that the Polβ/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.

  16. Selected Polymorphisms of Base Excision Repair Genes and Pancreatic Cancer Risk in Japanese

    PubMed Central

    Nakao, Makoto; Hosono, Satoyo; Ito, Hidemi; Watanabe, Miki; Mizuno, Nobumasa; Sato, Shigeki; Yatabe, Yasushi; Yamao, Kenji; Ueda, Ryuzo; Tajima, Kazuo; Tanaka, Hideo; Matsuo, Keitaro

    2012-01-01

    Background Although several reports have described a possible association between DNA repair genes and pancreatic cancer (PC) in smokers, this association has not been fully evaluated in an Asian population. We assessed the impact of genetic polymorphisms in the base excision repair (BER) pathway on PC risk among Japanese. Methods This case-control study compared the frequency of 5 single-nucleotide polymorphisms (SNPs) of BER genes, namely rs1052133 in OGG1, rs1799782 and rs25487 in XRCC1, rs1130409 in APE1, and rs1136410 in PARP1. SNPs were investigated using the TaqMan assay in 185 PC cases and 1465 controls. Associations of PC risk with genetic polymorphisms and gene–environment interaction were examined with an unconditional logistic regression model. Exposure to risk factors was assessed from the results of a self-administered questionnaire. We also performed haplotype-based analysis. Results We observed that the minor allele of rs25487 in XRCC1 was significantly associated with PC risk in the per-allele model (odds ratio = 1.29, CI = 1.01–1.65; trend P = 0.043). Haplotype analysis of XRCC1 also showed a statistically significant association with PC risk. No statistically significant interaction between XRCC1 polymorphisms and smoking status was seen. Conclusions Our findings suggest that XRCC1 polymorphisms affect PC risk in Japanese. PMID:22850545

  17. Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

    PubMed Central

    Goff, S P; Berg, P

    1978-01-01

    A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease. Images PMID:347445

  18. POLYMORPHISMS IN THE DNA NUCLEOTIDE EXCISION REPAIR GENES AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...

  19. Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks.

    PubMed

    Nakano, Toshiaki; Morishita, Soh; Katafuchi, Atsushi; Matsubara, Mayumi; Horikawa, Yusuke; Terato, Hiroaki; Salem, Amir M H; Izumi, Shunsuke; Pack, Seung Pil; Makino, Keisuke; Ide, Hiroshi

    2007-10-12

    DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

  20. Association of genetic polymorphism of the DNA base excision repair gene (APE-1 Asp/148 Glu) and HPV type (16/18) with the risk of cervix cancer in north Indian population.

    PubMed

    Shekari, Mohammad; Sobti, Ranbir Chander; Tamandani, Dor Mohammad Kordi; Malekzadeh, Keyanoosh; Kaur, Pushpinder; Suri, Vanita

    2008-01-01

    Cervical cancer is one of the most common neoplastic diseases affecting women, with a combined world wide incidence of almost half a million new cases. Reduced DNA repair capacity (DRC) can render a high risk of developing many types of cancer; including cervical cancer. Polymorphisms in DNA repair genes may contribute the genetic instability and carcinogenesis. Smoking experience and use of oral contraceptives have been confirmed to be risk factors for cervical cancer. The purpose of the present study was, therefore to investigate APE-1 genotypes (Asp/Asp, Asp/Glu, Glu/Glu) with different histological subtypes in cases compared with controls. It has been observed that Asp/Glu with Glu/Glu genotypes that combined we observed statistically significant with protective effect for developing of cervix cancer (OR-0.51, 95% CI 0.31-0.83, p-0.006). The combined Asp/Glu with Glu/Glu genotypes who were using oral contraceptives were shown to be statistically significant with reduced risk of cervical cancer (OR-0.22 95% CI- 0.11-0.47, p-0.0002). It has been suggested that significantly correlation between HPV 16 and users of oral contraceptives in certain APE-1 genotypes with reduced risk in developing cervix cancer. In conclusion we observed statistical significant association with reduced risk of cervix cancer in APE-1 with different genotypes, though, on the other hand, in association between HPV type 18 and those having SCC, highly increased risk of cervical cancer was observed.

  1. Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin.

    PubMed

    Herrero, Ana B; Martín-Castellanos, Cristina; Marco, Esther; Gago, Federico; Moreno, Sergio

    2006-08-15

    Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drug's cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drug's cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death.

  2. Mapping of genomic DNA loop organization in a 500-kilobase region of the Drosophila X chromosome by the topoisomerase II-mediated DNA loop excision protocol.

    PubMed Central

    Iarovaia, O; Hancock, R; Lagarkova, M; Miassod, R; Razin, S V

    1996-01-01

    The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA. PMID:8524309

  3. Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

    PubMed

    Wakasugi, Mitsuo; Sasaki, Takuma; Matsumoto, Megumi; Nagaoka, Miyuki; Inoue, Keiko; Inobe, Manabu; Horibata, Katsuyoshi; Tanaka, Kiyoji; Matsunaga, Tsukasa

    2014-10-10

    Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB.

  4. Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent HeLa cell extract.

    PubMed

    Petruseva, I O; Tikhanovich, I S; Maltseva, E A; Safronov, I V; Lavrik, O I

    2009-05-01

    Photoactivated DNA analogs of nucleotide excision repair (NER) substrates have been created that are 48-mer duplexes containing in internal positions pyrimidine nucleotides with bulky substituents imitating lesions. Fluorochloroazidopyridyl, anthracenyl, and pyrenyl groups introduced using spacer fragments at 4N and 5C positions of dCMP and dUMP were used as model damages. The gel retardation and photo-induced affinity modification techniques were used to study the interaction of modified DNA duplexes with proteins in HeLa cell extracts containing the main components of NER protein complexes. It is shown that the extract proteins selectively bind and form covalent adducts with the model DNA. The efficiency and selectivity of protein modification depend on the structure of used DNA duplex. Apparent molecular masses of extract proteins, undergoing modification, were estimated. Mutual influence of simultaneous presence of extract proteins and recombinant NER protein factors XPC-HR23B, XPA, and RPA on interaction with the model DNA was analyzed. The extract proteins and RPA competed for interaction with photoactive DNA, mutually decreasing the yield of modification products. In this case the presence of extract proteins at particular concentrations tripled the increase in yield of covalent adducts formed by XPC. It is supposed that the XPC subunit interaction with DNA is stimulated by endogenous HR23B present in the extract. Most likely, the mutual effect of XPA and extract proteins stimulating formation of covalent adducts with model DNA is due to the interaction of XPA with endogenous RPA of the extract. A technique based on the use of specific antibodies revealed that RPA present in the extract is a modification target for photoactive DNA imitating NER substrates.

  5. Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

    PubMed

    Overmeer, René M; Gourdin, Audrey M; Giglia-Mari, Ambra; Kool, Hanneke; Houtsmuller, Adriaan B; Siegal, Gregg; Fousteri, Maria I; Mullenders, Leon H F; Vermeulen, Wim

    2010-10-01

    Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

  6. Unplanned Excision of Extremity Soft Tissue Sarcoma in Korea: A Nationwide Study Based on a Claims Registry.

    PubMed

    Kang, Seungcheol; Kim, Han-Soo; Han, Ilkyu

    2015-01-01

    Unplanned excision of extremity soft tissue sarcoma (STS) is common and has detrimental effects not only on patients' oncologic outcomes but also on functional and economic issues. However, no study has analyzed a nationwide population-based database. To estimate the incidence and treatment pattern of unplanned excision in extremity STS in the Korean population, a nationwide epidemiologic study was performed using the Korean Health Insurance Review and Assessment Service database, a centralized nationwide healthcare claims registry of Korea that covers the entire Korean population. Among 1,517 patients with extremity STS in the 4-year study period, 553 (36.5%) underwent unplanned excision (unplanned group). About 80% of unplanned excisions were performed in tertiary or general hospitals. Of the unplanned group, 240 (43.4%) underwent re-excision with or without radiation therapy and/or chemotherapy, and 51 (9.2%) did not undergo re-excision but were treated with radiation therapy and/or chemotherapy; whereas, 262 (47.4%) did not undergo any further treatment following unplanned excision. This study is the first nationwide population-based study on the unplanned excision of extremity STS. The results may have implications in establishing preventive or therapeutic measures to reduce the burden of unplanned excision of extremity STS.

  7. Unplanned Excision of Extremity Soft Tissue Sarcoma in Korea: A Nationwide Study Based on a Claims Registry

    PubMed Central

    Kang, Seungcheol; Kim, Han-Soo; Han, Ilkyu

    2015-01-01

    Unplanned excision of extremity soft tissue sarcoma (STS) is common and has detrimental effects not only on patients’ oncologic outcomes but also on functional and economic issues. However, no study has analyzed a nationwide population-based database. To estimate the incidence and treatment pattern of unplanned excision in extremity STS in the Korean population, a nationwide epidemiologic study was performed using the Korean Health Insurance Review and Assessment Service database, a centralized nationwide healthcare claims registry of Korea that covers the entire Korean population. Among 1,517 patients with extremity STS in the 4-year study period, 553 (36.5%) underwent unplanned excision (unplanned group). About 80% of unplanned excisions were performed in tertiary or general hospitals. Of the unplanned group, 240 (43.4%) underwent re-excision with or without radiation therapy and/or chemotherapy, and 51 (9.2%) did not undergo re-excision but were treated with radiation therapy and/or chemotherapy; whereas, 262 (47.4%) did not undergo any further treatment following unplanned excision. This study is the first nationwide population-based study on the unplanned excision of extremity STS. The results may have implications in establishing preventive or therapeutic measures to reduce the burden of unplanned excision of extremity STS. PMID:26237049

  8. Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

    PubMed

    Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

    2014-06-01

    Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

  9. Germinal transmission of site-specific excised genomic DNA by the bacterial ParA resolvase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome engineering is an essential tool in research and product development. Behind some of the recent advances in plant gene transfer is the development of site-specific recombination systems that enable the precise manipulation of DNA, e.g. the deletion, integration or translocation of DNA. DNA ...

  10. Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice

    PubMed Central

    Skarpengland, Tonje; Holm, Sverre; Scheffler, Katja; Gregersen, Ida; Dahl, Tuva B.; Suganthan, Rajikala; Segers, Filip M.; Østlie, Ingunn; Otten, Jeroen J. T.; Luna, Luisa; Ketelhuth, Daniel F. J.; Lundberg, Anna M.; Neurauter, Christine G.; Hildrestrand, Gunn; Skjelland, Mona; Bjørndal, Bodil; Svardal, Asbjørn M.; Iversen, Per O.; Hedin, Ulf; Nygård, Ståle; Olstad, Ole K.; Krohg-Sørensen, Kirsten; Slupphaug, Geir; Eide, Lars; Kuśnierczyk, Anna; Folkersen, Lasse; Ueland, Thor; Berge, Rolf K.; Hansson, Göran K.; Biessen, Erik A. L.; Halvorsen, Bente; Bjørås, Magnar; Aukrust, Pål

    2016-01-01

    Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe−/−Neil3−/− mice on high-fat diet showed accelerated plaque formation as compared to Apoe−/− mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe−/−Neil3−/− mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage. PMID:27328939

  11. ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3.

    PubMed Central

    Weber, C A; Salazar, E P; Stewart, S A; Thompson, L H

    1990-01-01

    Human ERCC2 genomic clones give efficient, stable correction of the nucleotide excision repair defect in UV5 Chinese hamster ovary cells. One clone having a breakpoint just 5' of classical promoter elements corrects only transiently, implicating further flanking sequences in stable gene expression. The nucleotide sequences of a cDNA clone and genomic flanking regions were determined. The ERCC2 translated amino acid sequence has 52% identity (73% homology) with the yeast nucleotide excision repair protein RAD3. RAD3 is essential for cell viability and encodes a protein that is a single-stranded DNA dependent ATPase and an ATP dependent helicase. The similarity of ERCC2 and RAD3 suggests a role for ERCC2 in both cell viability and DNA repair and provides the first insight into the biochemical function of a mammalian nucleotide excision repair gene. Images Fig. 5. PMID:2184031

  12. Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases

    PubMed Central

    Woods, Ryan D.; O'Shea, Valerie L.; Chu, Aurea; Cao, Sheng; Richards, Jody L.; Horvath, Martin P.; David, Sheila S.

    2016-01-01

    MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for ‘retaining’ O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer. PMID:26673696

  13. Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases.

    PubMed

    Woods, Ryan D; O'Shea, Valerie L; Chu, Aurea; Cao, Sheng; Richards, Jody L; Horvath, Martin P; David, Sheila S

    2016-01-29

    MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer.

  14. Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH▿

    PubMed Central

    Zotter, Angelika; Luijsterburg, Martijn S.; Warmerdam, Daniël O.; Ibrahim, Shehu; Nigg, Alex; van Cappellen, Wiggert A.; Hoeijmakers, Jan H. J.; van Driel, Roel; Vermeulen, Wim; Houtsmuller, Adriaan B.

    2006-01-01

    The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process. PMID:17000769

  15. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

    SciTech Connect

    Gottlieb, J.; Muzyczka, N.

    1988-06-01

    When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

  16. Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

    PubMed

    Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

    2014-09-01

    A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

  17. Genetic Variation in Base Excision Repair Pathway Genes, Pesticide Exposure, and Prostate Cancer Risk

    PubMed Central

    Koutros, Stella; Berndt, Sonja I.; Andreotti, Gabriella; Hoppin, Jane A.; Sandler, Dale P.; Burdette, Laurie A.; Yeager, Meredith; Freeman, Laura E. Beane; Lubin, Jay H.; Ma, Xiaomei; Zheng, Tongzhang; Alavanja, Michael C.R.

    2011-01-01

    Background: Previous research indicates increased prostate cancer risk for pesticide applicators and pesticide manufacturing workers. Although underlying mechanisms are unknown, evidence suggests a role of oxidative DNA damage. Objectives: Because base excision repair (BER) is the predominant pathway involved in repairing oxidative damage, we evaluated interactions between 39 pesticides and 394 tag single-nucleotide polymorphisms (SNPs) for 31 BER genes among 776 prostate cancer cases and 1,444 male controls in a nested case–control study of white Agricultural Health Study (AHS) pesticide applicators. Methods: We used likelihood ratio tests from logistic regression models to determine p-values for interactions between three-level pesticide exposure variables (none/low/high) and SNPs (assuming a dominant model), and the false discovery rate (FDR) multiple comparison adjustment approach. Results: The interaction between fonofos and rs1983132 in NEIL3 [nei endonuclease VIII-like 3 (Escherichia coli)], which encodes a glycosylase that can initiate BER, was the most significant overall [interaction p-value (pinteract) = 9.3 × 10–6; FDR-adjusted p-value = 0.01]. Fonofos exposure was associated with a monotonic increase in prostate cancer risk among men with CT/TT genotypes for rs1983132 [odds ratios (95% confidence intervals) for low and high use compared with no use were 1.65 (0.91, 3.01) and 3.25 (1.78, 5.92), respectively], whereas fonofos was not associated with prostate cancer risk among men with the CC genotype. Carbofuran and S-ethyl dipropylthiocarbamate (EPTC) interacted similarly with rs1983132; however, these interactions did not meet an FDR < 0.2. Conclusions: Our significant finding regarding fonofos is consistent with previous AHS findings of increased prostate cancer risk with fonofos exposure among those with a family history of prostate cancer. Although requiring replication, our findings suggest a role of BER genetic variation in pesticide

  18. Interaction of Nucleotide Excision Repair Protein XPC-RAD23B with DNA Containing Benzo[a]pyrene-Derived Adduct and Apurinic/Apyrimidinic Site within a Cluster.

    PubMed

    Starostenko, L V; Maltseva, E A; Lebedeva, N A; Pestryakov, P E; Lavrik, O I; Rechkunova, N I

    2016-03-01

    The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms - NER and BER, respectively. Interaction of NER protein XPC-RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N(2)-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC-RAD23B crosslinks to DNA containing (+)-trans-BPDE-N(2)-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N(2)-dG isomers depends on the AP site position in the opposite strand. The influence of XPC-RAD23B on hydrolysis of an AP site clustered with BPDE-N(2)-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC-RAD23B was shown to stimulate the endonuclease and inhibit the 3'-5' exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage.

  19. Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation.

    PubMed

    Kode, Vasumathi; Mudd, Elisabeth A; Iamtham, Siriluck; Day, Anil

    2006-06-01

    We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aadA-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atpB promoter region. Efficient recombination between atpB repeats deletes the intervening foreign genes and 1,984 bp of plastid DNA (co-ordinates 57,424-59,317) containing the rbcL gene. Only five foreign bases are present in DeltarbcL plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbcL-related sequences in DeltarbcL plants indicated an extra-plastidic origin. Mutant DeltarbcL plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aadA-based transformation with the wild-type rbcL gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aadA genes. Rescue of the DeltarbcL mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbcL deletion mutant isolated here is readily transformed with the efficient aadA marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.

  20. AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP

    PubMed Central

    Hartl, Maximilian J.; Kretzschmar, Benedikt; Frohn, Anne; Nowrouzi, Ali; Rethwilm, Axel; Wöhrl, Birgitta M.

    2008-01-01

    Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher KM-values for polymerization but no change in KD-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency. PMID:18096624

  1. Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision.

    PubMed Central

    Osuna, R; Finkel, S E; Johnson, R C

    1991-01-01

    The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin-mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site-specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix-turn-helix DNA binding motif located near the Fis C-terminus (residues 74-93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N-terminal region reduce or abolish the stimulation of Hin-mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C-terminal DNA binding region that is required for promoting both DNA recombination reactions and an N-terminal region that is uniquely required for Hin-mediated inversion. Images PMID:1851089

  2. The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation

    PubMed Central

    Sun, H; He, L; Wu, H; Pan, F; Wu, X; Zhao, J; Hu, Z; Sekhar, C; Li, H; Zheng, L; Chen, H; Shen, B H; Guo, Z

    2017-01-01

    Flap endonuclease-1 (FEN1) is a multifunctional, structure-specific nuclease that has a critical role in maintaining human genome stability. FEN1 mutations have been detected in human cancer specimens and have been suggested to cause genomic instability and cancer predisposition. However, the exact relationship between FEN1 deficiency and cancer susceptibility remains unclear. In the current work, we report a novel colorectal cancer-associated FEN1 mutation, L209P. This mutant protein lacks the FEN, exonuclease (EXO) and gap endonuclease (GEN) activities of FEN1 but retains DNA-binding affinity. The L209P FEN1 variant interferes with the function of the wild-type FEN1 enzyme in a dominant-negative manner and impairs long-patch base excision repair in vitro and in vivo. Expression of L209P FEN1 sensitizes cells to DNA damage, resulting in endogenous genomic instability and cellular transformation, as well as tumor growth in a mouse xenograft model. These data indicate that human cancer-associated genetic alterations in the FEN1 gene can contribute substantially to cancer development. PMID:27270424

  3. Analysis of the base excision repair genes MTH1, OGG1 and MUTYH in patients with squamous oral carcinomas.

    PubMed

    Görgens, Heike; Müller, Annegret; Krüger, Stefan; Kuhlisch, Eberhard; König, Inke R; Ziegler, Andreas; Schackert, Hans K; Eckelt, Uwe

    2007-09-01

    A number of environmental factors, such as tobacco and alcohol, have been implicated, through oxidative DNA damage, in the development of squamous cell carcinomas of the head and neck (SCCHN). Several pathways are involved in the repair of DNA lesions caused by oxidative stress, such as the base excision repair system (BER), which repairs mutation involving 8-oxoguanine and comprises the MUTYH, OGG1 and MTH1 genes. We analysed 29 patients, assessing germline polymorphisms or mutations in these genes by complete genomic sequencing of exons and adjacent intronic regions. Thirty healthy blood donors served as controls. No pathogenic germline mutations were identified. We found common and rare new variants in the coding and adjacent intronic regions. In summary, our data do not support a major role for MUTYH, OGG1 and MTH1 variants in the etiology of sporadic squamous oral/oropharyngeal carcinomas. This does not exclude the involvement of the three BER genes in the tumorigenesis of SCCHN through other mechanisms such as promotor hypermethylation, genomic rearrangements or mutations involving regulatory sequences.

  4. Surprising repair activities of nonpolar analogs of 8-oxoG expose features of recognition and catalysis by base excision repair glycosylases.

    PubMed

    McKibbin, Paige L; Kobori, Akio; Taniguchi, Yosuke; Kool, Eric T; David, Sheila S

    2012-01-25

    Repair glycosylases locate and excise damaged bases from DNA, playing central roles in preservation of the genome and prevention of disease. Two key glycosylases, Fpg and hOGG1, function to remove the mutagenic oxidized base 8-oxoG (OG) from DNA. To investigate the relative contributions of conformational preferences, leaving group ability, enzyme-base hydrogen bonding, and nucleobase shape on damage recognition by these glycosylases, a series of four substituted indole nucleosides, based on the parent OG nonpolar isostere 2Cl-4F-indole, were tested as possible direct substrates of these enzymes in the context of 30 base pair duplexes paired with C. Surprisingly, single-turnover experiments revealed that Fpg-catalyzed base removal activity of two of the nonpolar analogs was superior to the native OG substrate. The hOGG1 glycosylase was also found to catalyze removal of three of the nonpolar analogs, albeit considerably less efficiently than removal of OG. Of note, the analog that was completely resistant to hOGG1-catalyzed excision has a chloro-substituent at the position of NH7 of OG, implicating the importance of recognition of this position in catalysis. Both hOGG1 and Fpg retained high affinity for the duplexes containing the nonpolar isosteres. These studies show that hydrogen bonds between base and enzyme are not needed for efficient damage recognition and repair by Fpg and underscore the importance of facile extrusion from the helix in its damaged base selection. In contrast, damage removal by hOGG1 is sensitive to both hydrogen bonding groups and nucleobase shape. The relative rates of excision of the analogs with the two glycosylases highlight key differences in their mechanisms of damaged base recognition and removal.

  5. Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.

    PubMed Central

    Park, E M; Shigenaga, M K; Degan, P; Korn, T S; Kitzler, J W; Wehr, C M; Kolachana, P; Ames, B N

    1992-01-01

    An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day. PMID:1565629

  6. Repair of DNA-protein cross-links in an excision repair-deficient human cell line and its simian virus 40-transformed derivative

    SciTech Connect

    Gantt, R.; Taylor, W.G.; Camalier, R.F.; Stephens, E.V.

    1984-05-01

    DNA-protein cross-links are induced in mammalian cells by X-rays, ultraviolet light, fluorescent light, and numerous chemical carcinogens. Others have shown that these cross-links are repaired by normal cells but that excision repair-deficient xeroderma pigmentosum (XP) Group A cells, XP12BE, are deficient in repair of these bulky adducts. This paper compares the DNA-protein cross-link repair competency of another XP Group A strain, XP20S, with its more rapidly proliferating simian virus 40-transformed derivative line and with normal human skin fibroblasts. DNA-protein cross-links were induced with 20 microM transplatinum(II)diamminedichloride and assayed by the membrane alkaline elution procedure of Kohn. Treated and untreated cells are lysed on a polycarbonate membrane filter, and the coelution rates of the DNA at pH 12.2 are compared; DNA-protein cross-links retard elution of DNA. The repair competency of XP20S cells for trans-platinum(II)diamminedichloride-induced DNA-protein cross-links was similar to that of XP12BE cells, but the competency of the simian virus 40-transformed XP20S cells was nearly equal to that of normal human skin fibroblasts. These results suggest that either cell cycling compensates for the genetic deficiency present in the nucleotide excision process of XP Group A cells or that a process other than nucleotide excision can repair these lesions; this process requires cell cycling or activation by the virus.

  7. Low risk of contamination with automated and manual excision of dried blood spots for HIV DNA PCR testing in the routine laboratory.

    PubMed

    Driver, Glenn A; Patton, Janet C; Moloi, Jackie; Stevens, Wendy S; Sherman, Gayle G

    2007-12-01

    In low resource settings the inability to diagnose HIV in infants early presents a major obstacle to providing care for HIV-infected children. Dried blood spot samples offer a solution to the scarcity of skills available for venesecting young infants but pose challenges to laboratories because their processing requirements are distinct from that of the liquid blood samples widely used for viral detection assays. Different methods of excising 287 paired HIV-positive and HIV-negative dried blood spot samples (n=574) for testing by the Roche Amplicor HIV-1 DNA assay version 1.5 were assessed. A manual punch in conjunction with three different cleaning protocols (n=372) and an automated punch (BSD 1000 GenePunch) using a single cleaning protocol (n=202) was assessed for the risk of cross contamination between samples. A single false positive result obtained using the automated punch may have been attributable to contamination during the excision step of the assay. Excision of dried blood spot samples is associated with a very low risk of cross contamination regardless of the instrument or cleaning intervention used. The process of excising dried blood spot samples should not compromise the results of HIV viral detection assays performed on dried blood spots in routine laboratories which is encouraging for increasing access to an accurate diagnosis of HIV in infants.

  8. Iterative marker excision system.

    PubMed

    Myronovskyi, Maksym; Rosenkränzer, Birgit; Luzhetskyy, Andriy

    2014-05-01

    The deletions of large genomic DNA fragments and consecutive gene knockouts are prerequisites for the generation of organisms with improved properties. One of the key issues in this context is the removal of antibiotic resistance markers from engineered organisms without leaving an active recombinase recognition site. Here, we report the establishment of an iterative marker excision system (IMES) that solves this problem. Based on the phiC31 integrase and its mutant att sites, IMES can be used for highly effective deletion of DNA fragments between inversely oriented B-CC and P-GG sites. The B-CC and P-GG sites are derived from attB and attP by substitution of the central core TT dinucleotide with CC and GG, respectively. An unnatural RR site that resides in the chromosome following deletion is the joining product of the right shoulders of B-CC and P-GG. We show that the RR sites do not recombine with each other as well as the RR site recombines with B-CC. The recombination efficiencies between RR and P-GG or RR and LL are only 0.1 % and 1 %, respectively. Thus, IMES can be used for multistep genomic engineering without risking unwanted DNA recombination. The fabrication of multi-purpose antibiotic cassettes and examples of the utilisation of IMES are described.

  9. Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivo.

    PubMed

    Tremblay, Maxime; Toussaint, Martin; D'Amours, Annie; Conconi, Antonio

    2009-02-01

    The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.

  10. Differential survival of Escherichia coli uvrA, uvrB, and uvrC mutants to psoralen plus UV-A (PUVA): Evidence for uncoupled action of nucleotide excision repair to process DNA adducts.

    PubMed

    Lage, Claudia; Gonçalves, Silvia R F; Souza, Luciana L; de Pádula, Marcelo; Leitão, Alvaro C

    2010-01-21

    The nucleotide excision repair mechanism (NER) of Escherichia coli is responsible for the recognition and elimination of more than twenty different DNA lesions. Herein, we evaluated the in vivo role of NER in the repair of DNA adducts generated by psoralens (mono- or bi-functional) and UV-A light (PUVA) in E. coli. Cultures of wild-type E. coli K12 and mutants for uvrA, uvrB, uvrC or uvrAC genes were treated with PUVA and cell survival was determined. In parallel, kinetics of DNA repair was also evaluated by the comparison of DNA sedimentation profiles in all the strains after PUVA treatment. The uvrB mutant was more sensitive to PUVA treatment than all the other uvr mutant strains. Wild-type strain, and uvrA and uvrC mutants were able to repair PUVA-induced lesions, as seen by DNA sedimentation profiles, while the uvrB mutant was unable to repair the lesions. In addition, a quadruple fpg nth xth nfo mutant was unable to nick PUVA-treated DNA when the crude cell-free extract was used to perform plasmid nicking. These data suggest that DNA repair of PUVA-induced lesions may require base excision repair functions, despite proficient UvrABC activity. These results point to a specific role for UvrB protein in the repair of psoralen adducts, which appear to be independent of UvrA or UvrC proteins, as described for the classical UvrABC endonuclease mechanism.

  11. DNA based molecular motors

    NASA Astrophysics Data System (ADS)

    Michaelis, Jens; Muschielok, Adam; Andrecka, Joanna; Kügel, Wolfgang; Moffitt, Jeffrey R.

    2009-12-01

    Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid-a process that is driven by one of the most powerful molecular motors.

  12. In vitro Repair of Oxidative DNA Damage by Human Nucleotide Excision Repair System: Possible Explanation for Neurodegeneration in Xeroderma Pigmentosum Patients

    NASA Astrophysics Data System (ADS)

    Reardon, Joyce T.; Bessho, Tadayoshi; Kung, Hsiang Chuan; Bolton, Philip H.; Sancar, Aziz

    1997-08-01

    Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

  13. Laboratory technology for population-based screening for severe combined immunodeficiency in neonates: the winner is T-cell receptor excision circles.

    PubMed

    Puck, Jennifer M

    2012-03-01

    The most profound primary immunodeficiency disease, severe combined immunodeficiency (SCID), is fatal in infancy unless affected infants are provided with an adaptive immune system through allogeneic hematopoietic cell transplantation, enzyme replacement, or gene therapy. However, most infants with SCID lack a family history or any clinical clues before the onset of infections, making this serious but treatable disease a candidate for population-based newborn screening. Of several approaches considered for SCID screening, testing for T-cell receptor excision circles (TRECs), a DNA biomarker of normal T-cell development, has proved successful. TREC numbers can be measured in DNA isolated from the dried bloodspots already routinely collected for newborn screening. Infants with low or absent TRECs can thus be identified and referred for confirmatory testing and prompt intervention. TREC testing of newborns is now being performed in several states, indicating that this addition to the newborn screening panel can be successfully integrated into state public health programs.

  14. Oxidative stress alters base excision repair pathway and increases apoptotic response in Apurinic/apyrimidinic endonuclease 1/Redox factor-1 haploinsufficient mice

    PubMed Central

    Unnikrishnan, Archana; Raffoul, Julian J.; Patel, Hiral V.; Prychitko, Thomas M.; Anyangwe, Njwen; Meira, Lisiane B.; Friedberg, Errol C.; Cabelof, Diane C.; Heydari, Ahmad R.

    2009-01-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-κB, and the major 5’-endonuclease in base excision repair (BER). We utilized mice containing heterozygous gene-targeted deletion of APE1/Ref-1 (Apex+/-) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-κB DNA binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress where a significant increase in GADD45g expression, p53 protein stability and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UDG) and bifunctional (OGG1) DNA glycosylase initiated BER in liver of Apex+/- mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), while removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex+/- mice exposed to 2-NP displayed a significant decline in 3’-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase initiated BER. PMID:19268524

  15. Chitosan-based copper nanocomposite accelerates healing in excision wound model in rats.

    PubMed

    Gopal, Anu; Kant, Vinay; Gopalakrishnan, Anu; Tandan, Surendra K; Kumar, Dinesh

    2014-05-15

    Copper possesses efficacy in wound healing which is a complex phenomenon involving various cells, cytokines and growth factors. Copper nanoparticles modulate cells, cytokines and growth factors involved in wound healing in a better way than copper ions. Chitosan has been shown to be beneficial in healing because of its antibacterial, antifungal, biocompatible and biodegradable polymeric nature. In the present study, chitosan-based copper nanocomposite (CCNC) was prepared by mixing chitosan and copper nanoparticles. CCNC was applied topically to evaluate its wound healing potential and to study its effects on some important components of healing process in open excision wound model in adult Wistar rats. Significant increase in wound contraction was observed in the CCNC-treated rats. The up-regulation of vascular endothelial growth factor (VEGF) and transforming growth factor-beta1(TGF-β1) by CCNC-treatment revealed its role in facilitating angiogenesis, fibroblast proliferation and collagen deposition. The tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were significantly decreased and increased, respectively, in CCNC-treated rats. Histological evaluation showed more fibroblast proliferation, collagen deposition and intact re-epithelialization in CCNC-treated rats. Immunohistochemistry of CD31 revealed marked increase in angiogenesis. Thus, we concluded that chitosan-based copper nanocomposite efficiently enhanced cutaneous wound healing by modulation of various cells, cytokines and growth factors during different phases of healing process.

  16. Activation of GLP-1 Receptor Enhances Neuronal Base Excision Repair via PI3K-AKT-Induced Expression of Apurinic/Apyrimidinic Endonuclease 1

    PubMed Central

    Yang, Jenq-Lin; Chen, Wei-Yu; Chen, Yin-Ping; Kuo, Chao-Ying; Chen, Shang-Der

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that increases cellular glucose up-take to decrease blood sugar. Recent studies, however, suggest that the function of GLP-1 is not only to decrease blood sugar, but also acts as a neurotrophic factor that plays a role in neuronal survival, neurite outgrowth, and protects synaptic plasticity and memory formation from effects of β-amyloid. Oxidative DNA damage occurs during normal neuron-activity and in many neurological diseases. Our study describes how GLP-1 affected the ability of neurons to ameliorate oxidative DNA damage. We show that activation of GLP-1 receptor (GLP-1R) protect cortical neurons from menadione induced oxidative DNA damage via a signaling pathway involving enhanced DNA repair. GLP-1 stimulates DNA repair by activating the cyclic AMP response element binding protein (CREB) which, consequently, induces the expression of apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision DNA repair (BER) pathway. In this study, APE1 expression was down-regulated as a consequence phosphatidylinositol-3 kinase (PI3K) suppression by the inhibitor LY294002, but not by the suppression of MEK activity. Ischemic stroke is typically caused by overwhelming oxidative-stress in brain cells. Administration of exentin-4, an analogue of GLP-1, efficiently enhanced DNA repair in brain cells of ischemic stroke rats. Our study suggests that a new function of GLP-1 is to elevate DNA repair by inducing the expression of the DNA repair protein APE1. PMID:27698937

  17. Using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro.

    PubMed

    Córdoba-Cañero, Dolores; Roldán-Arjona, Teresa; Ariza, Rafael R

    2012-01-01

    Base excision repair (BER) is a major pathway for the removal of endogenous and exogenous DNA damage. This repair mechanism is initiated by DNA glycosylases that excise the altered base, and continues through alternative routes that culminate in DNA resynthesis and ligation. In contrast to the information available for microbes and animals, our knowledge about this important DNA repair pathway in plants is very limited, partially due to a lack of biochemical approaches. Here we describe an in vitro assay to monitor BER in cell-free extracts from the model plant Arabidopsis thaliana. The assay uses labeled DNA substrates containing a single damaged base within a restriction site, and allows detection of fully repaired molecules as well as DNA repair intermediates. The method is easily applied to measure the repair activity of purified proteins and can be successfully used in combination with the extensive array of biological resources available for Arabidopsis.

  18. Deficiency of base excision repair enzyme NEIL3 drives increased predisposition to autoimmunity

    PubMed Central

    Massaad, Michel J.; Zhou, Jia; Tsuchimoto, Daisuke; Chou, Janet; Jabara, Haifa; Janssen, Erin; Glauzy, Salomé; Olson, Brennan G.; Morbach, Henner; Ohsumi, Toshiro K.; Schmitz, Klaus; Kane, Jennifer; Torisu, Kumiko; Chouery, Eliane; Megarbane, Andre; Kang, Peter B.; Al-Idrissi, Eman; Aldhekri, Hasan; Meffre, Eric; Mizui, Masayuki; Manis, John P.; Al-Herz, Waleed; Wallace, Susan S.; Geha, Raif S.

    2016-01-01

    Alterations in the apoptosis of immune cells have been associated with autoimmunity. Here, we have identified a homozygous missense mutation in the gene encoding the base excision repair enzyme Nei endonuclease VIII-like 3 (NEIL3) that abolished enzymatic activity in 3 siblings from a consanguineous family. The NEIL3 mutation was associated with fatal recurrent infections, severe autoimmunity, hypogammaglobulinemia, and impaired B cell function in these individuals. The same homozygous NEIL3 mutation was also identified in an asymptomatic individual who exhibited elevated levels of serum autoantibodies and defective peripheral B cell tolerance, but normal B cell function. Further analysis of the patients revealed an absence of LPS-responsive beige-like anchor (LRBA) protein expression, a known cause of immunodeficiency. We next examined the contribution of NEIL3 to the maintenance of self-tolerance in Neil3–/– mice. Although Neil3–/– mice displayed normal B cell function, they exhibited elevated serum levels of autoantibodies and developed nephritis following treatment with poly(I:C) to mimic microbial stimulation. In Neil3–/– mice, splenic T and B cells as well as germinal center B cells from Peyer’s patches showed marked increases in apoptosis and cell death, indicating the potential release of self-antigens that favor autoimmunity. These findings demonstrate that deficiency in NEIL3 is associated with increased lymphocyte apoptosis, autoantibodies, and predisposition to autoimmunity. PMID:27760045

  19. Importance of ligand structure in DNA/protein binding, mutagenicity, excision repair and nutritional aspects of chromium(III) complexes.

    PubMed

    Vaidyanathan, V G; Asthana, Yamini; Nair, Balachandran Unni

    2013-02-21

    Chromium is extensively used in leather, chrome plating and refining industries. On one hand the occupational exposure to chromium leads to cancer, whereas on the contrary certain Cr(III) compounds have been proposed as nutritional supplements for Type II diabetes and as muscle building agents. Despite the positive outlook of chromium as a bio-essential element, there is increasing concern over the therapeutic application of Cr(III) based supplements, its bioavailability and toxicity profile. In this perspective, we discuss the role of ligand structure in mediating the interaction of chromium(III) complexes with DNA/protein, their mutagenic outcomes, adduct reparability and as nutritional supplements.

  20. Excision efficiency is not strongly coupled to transgenic rate: cell type-dependent transposition efficiency of sleeping beauty and piggyBac DNA transposons.

    PubMed

    Kolacsek, Orsolya; Erdei, Zsuzsa; Apáti, Agota; Sándor, Sára; Izsvák, Zsuzsanna; Ivics, Zoltán; Sarkadi, Balázs; Orbán, Tamás I

    2014-08-01

    The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, "overproduction inhibition," a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in-house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.

  1. Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency of Sleeping Beauty and piggyBac DNA Transposons

    PubMed Central

    Kolacsek, Orsolya; Erdei, Zsuzsa; Apáti, Ágota; Sándor, Sára; Izsvák, Zsuzsanna; Ivics, Zoltán; Sarkadi, Balázs

    2014-01-01

    Abstract The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, “overproduction inhibition,” a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own “in-house” platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2–3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future. PMID:25045962

  2. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes.

    PubMed

    Piao, Mei Jing; Hewage, Susara Ruwan Kumara Madduma; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Lee, Nam Ho; Hyun, Jin Won

    2015-09-02

    We investigated the protective properties of diphlorethohydroxycarmalol (DPHC), a phlorotannin, against ultraviolet B (UVB) radiation-induced cyclobutane pyrimidine dimers (CPDs) in HaCaT human keratinocytes. The nucleotide excision repair (NER) system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV) radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementing 1 (ERCC1), which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1) and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system.

  3. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes

    PubMed Central

    Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Lee, Nam Ho; Hyun, Jin Won

    2015-01-01

    We investigated the protective properties of diphlorethohydroxycarmalol (DPHC), a phlorotannin, against ultraviolet B (UVB) radiation-induced cyclobutane pyrimidine dimers (CPDs) in HaCaT human keratinocytes. The nucleotide excision repair (NER) system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV) radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementing 1 (ERCC1), which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1) and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system. PMID:26404324

  4. The Late Developmental Pattern of Mu Transposon Excision Is Conferred by a Cauliflower Mosaic Virus 35S –Driven MURA cDNA in Transgenic Maize

    PubMed Central

    Raizada, Manish N.; Walbot, Virginia

    2000-01-01

    The MuDR element responsible for Mutator activities in maize encodes two genes, mudrA and mudrB. Each encodes multiple transcripts hypothesized to regulate, directly or indirectly, the unique late timing and switch in transposition mechanism during maize development. mudrA, which encodes the MURA transposase, is unstable in bacterial plasmids, a technical problem solved by using phage M13 as a vector to prepare DNA for biolistic transformation. In transgenic maize, a single 2.7-kb mudrA cDNA predicted to encode an 823–amino acid protein is sufficient to catalyze late somatic excisions, despite removal of the native promoter, alternative transcription start sites, known introns, polymorphic 5′ and 3′ untranslated sequences, and the mudrB gene. These results suggest that post-translational regulation confers Mu excision timing. The transgene is active in lines containing silencing MuDR elements. This suggests that endogenous MuDR transposons do not measurably immunize the host against expression of a homologous transgene. PMID:10634904

  5. Terahertz transmission vs reflection imaging and model-based characterization for excised breast carcinomas

    PubMed Central

    Bowman, Tyler; El-Shenawee, Magda; Campbell, Lucas K.

    2016-01-01

    This work presents experimental and analytical comparison of terahertz transmission and reflection imaging modes for assessing breast carcinoma in excised paraffin-embedded human breast tissue. Modeling for both transmission and reflection imaging is developed. The refractive index and absorption coefficient of the tissue samples are obtained. The reflection measurements taken at the system’s fixed oblique angle of 30° are shown to be a hybridization of TE and TM modes. The models are validated with transmission spectroscopy at fixed points on fresh bovine muscle and fat tissues. Images based on the calculated absorption coefficient and index of refraction of bovine tissue are successfully compared with the terahertz magnitude and phase measured in the reflection mode. The validated techniques are extended to 20 and 30 μm slices of fixed human lobular carcinoma and infiltrating ductal carcinoma mounted on polystyrene microscope slides in order to investigate the terahertz differentiation of the carcinoma with non-cancerous tissue. Both transmission and reflection imaging show clear differentiation in carcinoma versus healthy tissue. However, when using the reflection mode, in the calculation of the thin tissue properties, the absorption is shown to be sensitive to small phase variations that arise due to deviations in slide and tissue thickness and non-ideal tissue adhesion. On the other hand, the results show that the transmission mode is much less sensitive to these phase variations. The results also demonstrate that reflection imaging provides higher resolution and more clear margins between cancerous and fibroglandular regions, cancerous and fatty regions, and fibroglandular and fatty tissue regions. In addition, more features consistent with high power pathology images are exhibited in the reflection mode images. PMID:27699136

  6. Terahertz transmission vs reflection imaging and model-based characterization for excised breast carcinomas.

    PubMed

    Bowman, Tyler; El-Shenawee, Magda; Campbell, Lucas K

    2016-09-01

    This work presents experimental and analytical comparison of terahertz transmission and reflection imaging modes for assessing breast carcinoma in excised paraffin-embedded human breast tissue. Modeling for both transmission and reflection imaging is developed. The refractive index and absorption coefficient of the tissue samples are obtained. The reflection measurements taken at the system's fixed oblique angle of 30° are shown to be a hybridization of TE and TM modes. The models are validated with transmission spectroscopy at fixed points on fresh bovine muscle and fat tissues. Images based on the calculated absorption coefficient and index of refraction of bovine tissue are successfully compared with the terahertz magnitude and phase measured in the reflection mode. The validated techniques are extended to 20 and 30 μm slices of fixed human lobular carcinoma and infiltrating ductal carcinoma mounted on polystyrene microscope slides in order to investigate the terahertz differentiation of the carcinoma with non-cancerous tissue. Both transmission and reflection imaging show clear differentiation in carcinoma versus healthy tissue. However, when using the reflection mode, in the calculation of the thin tissue properties, the absorption is shown to be sensitive to small phase variations that arise due to deviations in slide and tissue thickness and non-ideal tissue adhesion. On the other hand, the results show that the transmission mode is much less sensitive to these phase variations. The results also demonstrate that reflection imaging provides higher resolution and more clear margins between cancerous and fibroglandular regions, cancerous and fatty regions, and fibroglandular and fatty tissue regions. In addition, more features consistent with high power pathology images are exhibited in the reflection mode images.

  7. Small-molecule inhibitors of proteins involved in base excision repair potentiate the anti-tumorigenic effect of existing chemotherapeutics and irradiation

    PubMed Central

    Reed, April M; Fishel, Melissa L; Kelley, Mark R

    2009-01-01

    There has been a recent upsurge in the development of small-molecule inhibitors specific to DNA repair proteins or proteins peripherally involved in base excision repair and the DNA damage response. These specific, nominally toxic inhibitors are able to potentiate the effect of existing cancer cell treatments in a wide array of cancers. One of the largest obstacles to overcome in the treatment of cancer is incomplete killing with initial cancer treatments, leading to resistant cancer. The progression of our understanding of cancer and normal cell responses to DNA damage has allowed us to develop biomarkers that we can use to help us predict responses of cancers, more specifically target cancer cells and overcome resistance. Initial successes using these small-molecule DNA repair inhibitors in target-validation experiments and in the early stages of clinical trials indicate an important role for these inhibitors, and allow for the possibility of a future in which cancers are potentially treated in a highly specific, individual manner. PMID:19519210

  8. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

    PubMed Central

    MacInnes, M A; Dickson, J A; Hernandez, R R; Learmonth, D; Lin, G Y; Mudgett, J S; Park, M S; Schauer, S; Reynolds, R J; Strniste, G F

    1993-01-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Our available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeasts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. Images PMID:8413238

  9. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of saccharomyces cerevisiae and schizosaccharomyces pombe

    SciTech Connect

    MacInnes, M.A.; Dickson, J.A.; Hernandez, R.R.; Lin, G.Y.; Park, M.S.; Schauer, S.; Reynolds, R.J.; Strniste, G.F. ); Learmonth, D. ); Mudgett, J.S. ); Yu, J.Y. )

    1993-10-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. The authors have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Their available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5[prime]-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Their evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed for several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeast, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Their analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. 69 refs., 6 figs., 1 tab.

  10. The nucleotide excision repair system of Borrelia burgdorferi is the sole pathway involved in repair of DNA damage by UV light.

    PubMed

    Hardy, Pierre-Olivier; Chaconas, George

    2013-05-01

    To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival of B. burgdorferi exposed to UV light. However, the disruption of the B. burgdorferi nucleotide excision repair (NER) pathway (uvrA, uvrB, uvrC, and uvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required for B. burgdorferi resistance to nitrosative damage. Finally, disruption of uvrA, uvrC, and uvrD had only a minor effect upon murine infection by increasing the time required for dissemination.

  11. Cinnamate-based DNA photolithography

    NASA Astrophysics Data System (ADS)

    Feng, Lang; Romulus, Joy; Li, Minfeng; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin; Seeman, Nadrian C.; Weck, Marcus; Chaikin, Paul

    2013-08-01

    As demonstrated by means of DNA nanoconstructs, as well as DNA functionalization of nanoparticles and micrometre-scale colloids, complex self-assembly processes require components to associate with particular partners in a programmable fashion. In many cases the reversibility of the interactions between complementary DNA sequences is an advantage. However, permanently bonding some or all of the complementary pairs may allow for flexibility in design and construction. Here, we show that the substitution of a cinnamate group for a pair of complementary bases provides an efficient, addressable, ultraviolet light-based method to bond complementary DNA covalently. To show the potential of this approach, we wrote micrometre-scale patterns on a surface using ultraviolet light and demonstrated the reversible attachment of conjugated DNA and DNA-coated colloids. Our strategy enables both functional DNA photolithography and multistep, specific binding in self-assembly processes.

  12. DNA-based soft phases.

    PubMed

    Bellini, Tommaso; Cerbino, Roberto; Zanchetta, Giuliano

    2012-01-01

    This chapter reviews the state-of-the-art in the study of molecular or colloidal systems whose mutual interactions are mediated by DNA molecules. In the last decade, the robust current knowledge of DNA interactions has enabled an impressive growth of self-assembled DNA-based structures that depend crucially on the properties of DNA-DNA interactions. In many cases, structures are built on design by exploiting the programmable selectivity of DNA interactions and the modularity of their strength. The study of DNA-based materials is definitely an emerging field in condensed matter physics, nanotechnology, and material science. This chapter will consider both systems that are entirely constructed by DNA and hybrid systems in which latex or metal colloidal particles are coated by DNA strands. We will confine our discussion to systems in which DNA-mediated interactions promote the formation of "phases," that is structures extending on length scales much larger than the building blocks. Their self-assembly typically involves a large number of interacting particles and often features hierarchical stages of structuring. Because of the possibility of fine-tuning the geometry and strength of the DNA-mediated interactions, these systems are characterized by a wide variety of patterns of self-assembly, ranging from amorphous, to liquid crystalline, to crystalline in one, two, or three dimensions.

  13. Variation in PAH-related DNA adduct levels among non-smokers: the role of multiple genetic polymorphisms and nucleotide excision repair phenotype

    PubMed Central

    Etemadi, Arash; Islami, Farhad; Phillips, David H.; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T; Taylor, Philip R; Boffetta, Paolo; Abnet, Christian C; Dawsey, Sanford M; Malekzadeh, Reza; van Schooten, Frederik J.

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly-selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to xenobiotic metabolism and 12 SNPs in 8 DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by 32P-postlabelling. Multivariable regression models were compared by Akaike’s information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 108 nucleotides (mean: 5.8±3.1). DNA adduct level was significantly lower in homozygotes for NAT2 slow alleles and ERCC5 non risk-allele genotype, and was higher in the MPO homozygote risk-allele genotype. The sum of risk alleles in these genes significantly correlated with the log-adduct level (r=0.4, p<0.001). Compared with the environmental model, adding phase I SNPs and NER capacity provided the best fit, and could explain 17% more of the variation in adduct levels. NER capacity was affected by polymorphisms in the MTHFR and ERCC1 genes. Female non-smokers in this population had PAH-related DNA adduct levels 3-4 times higher than smokers and occupationally-exposed groups in previous studies, with large inter-individual variation which could best be explained by a combination of phase I genes and NER capacity. PMID:23175176

  14. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

    PubMed

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

  15. Low-intensity red and infrared lasers affect mRNA expression of DNA nucleotide excision repair in skin and muscle tissue.

    PubMed

    Sergio, Luiz Philippe S; Campos, Vera Maria A; Vicentini, Solange C; Mencalha, Andre Luiz; de Paoli, Flavia; Fonseca, Adenilson S

    2016-04-01

    Lasers emit light beams with specific characteristics, in which wavelength, frequency, power, fluence, and emission mode properties determine the photophysical, photochemical, and photobiological responses. Low-intensity lasers could induce free radical generation in biological tissues and cause alterations in macromolecules, such as DNA. Thus, the aim of this work was to evaluate excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2 (ERCC2) messenger RNA (mRNA) expression in biological tissues exposed to low-intensity lasers. Wistar rat (n = 28, 4 for each group) skin and muscle were exposed to low-intensity red (660 nm) and near-infrared (880 nm) lasers at different fluences (25, 50, and 100 J/cm(2)), and samples of these tissues were withdrawn for RNA extraction, cDNA synthesis, and gene expression evaluation by quantitative polymerase chain reaction. Laser exposure was in continuous wave and power of 100 mW. Data show that ERCC1 and ERCC2 mRNA expressions decrease in skin (p < 0.001) exposed to near-infrared laser, but increase in muscle tissue (p < 0.001). ERCC1 mRNA expression does not alter (p > 0.05), but ERCC2 mRNA expression decreases in skin (p < 0.001) and increases in muscle tissue (p < 0.001) exposed to red laser. Our results show that ERCC1 and ERCC2 mRNA expression is differently altered in skin and muscle tissue exposed to low-intensity lasers depending on wavelengths and fluences used in therapeutic protocols.

  16. DNA-based hybrid catalysis.

    PubMed

    Rioz-Martínez, Ana; Roelfes, Gerard

    2015-04-01

    In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed.

  17. A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair

    PubMed Central

    Ranes, Michael; Boeing, Stefan; Wang, Yuming; Wienholz, Franziska; Menoni, Hervé; Walker, Jane; Encheva, Vesela; Chakravarty, Probir; Mari, Pierre-Olivier; Stewart, Aengus; Giglia-Mari, Giuseppina; Snijders, Ambrosius P.; Vermeulen, Wim; Svejstrup, Jesper Q.

    2016-01-01

    Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein. PMID:27060134

  18. The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1.

    PubMed

    Das, Aditi; Boldogh, Istvan; Lee, Jae Wan; Harrigan, Jeanine A; Hegde, Muralidhar L; Piotrowski, Jason; de Souza Pinto, Nadja; Ramos, William; Greenberg, Marc M; Hazra, Tapas K; Mitra, Sankar; Bohr, Vilhelm A

    2007-09-07

    The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.

  19. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  20. Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

    PubMed Central

    Naumovski, L; Friedberg, E C

    1982-01-01

    We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size. Images PMID:6749808

  1. DNA Fragments Assembly Based on Nicking Enzyme System

    PubMed Central

    Wang, Rui-Yan; Shi, Zhen-Yu; Guo, Ying-Ying; Chen, Jin-Chun; Chen, Guo-Qiang

    2013-01-01

    A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3′-end or 5′-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB). PMID:23483947

  2. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process.

  3. Nucleotide excision repair in humans

    PubMed Central

    Spivak, Graciela

    2015-01-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  4. Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

    PubMed Central

    Liu, Geyi; Aronovich, Elena L.; Cui, Zongbin; Whitley, Chester B.; Hackett, Perry B.

    2007-01-01

    A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase–transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. PMID:15133768

  5. The Shu complex promotes error-free tolerance of alkylation-induced base excision repair products

    PubMed Central

    Godin, Stephen K.; Zhang, Zhuying; Herken, Benjamin W.; Westmoreland, James W.; Lee, Alison G.; Mihalevic, Michael J.; Yu, Zhongxun; Sobol, Robert W.; Resnick, Michael A.; Bernstein, Kara A.

    2016-01-01

    Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion. PMID:27298254

  6. Principles of DNA architectonics: design of DNA-based nanoobjects

    NASA Astrophysics Data System (ADS)

    Vinogradova, O. A.; Pyshnyi, D. V.

    2012-02-01

    The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

  7. Visualizing the search for radiation-damaged DNA bases in real time

    NASA Astrophysics Data System (ADS)

    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.

  8. Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

    PubMed

    Maira, N; Torres, T M; de Oliveira, A L; de Medeiros, S R B; Agnez-Lima, L F; Lima, J P M S; Scortecci, K C

    2014-05-01

    Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

  9. The MUTYH base excision repair gene protects against inflammation-associated colorectal carcinogenesis

    PubMed Central

    Grasso, Francesca; Di Meo, Serena; De Luca, Gabriele; Pasquini, Luca; Rossi, Stefania; Boirivant, Monica; Biffoni, Mauro; Bignami, Margherita; Di Carlo, Emma

    2015-01-01

    MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). Mutyh−/− mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in Mutyh−/− than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated Mutyh−/− mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFNγ, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from Mutyh−/− mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-β-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in Mutyh−/− mice provides a good model for MAP. PMID:26109431

  10. Polymorphisms in nucleotide excision repair genes, smoking and breast cancer in African Americans and whites: a population-based case-control study.

    PubMed

    Mechanic, Leah E; Millikan, Robert C; Player, Jon; de Cotret, Allan René; Winkel, Scott; Worley, Kendra; Heard, Kristin; Heard, Kimberley; Tse, Chiu-Kit; Keku, Temitope

    2006-07-01

    Polymorphisms exist in several genes involved in nucleotide excision repair (NER), the principal pathway for removal of smoking-induced DNA damage. An epidemiologic study was conducted to determine whether these polymorphisms modify the association between smoking and breast cancer. DNA samples and exposure histories were analyzed as part of a large population-based case-control study of breast cancer in North Carolina. The study population included 2311 cases (894 African Americans, 1417 whites) and 2022 controls (788 African Americans, 1234 whites). Odds ratios (ORs) were calculated for breast cancer and smoking, and for breast cancer and nine non-synonymous coding polymorphisms in six NER genes (XPD codons 312 and 751, RAD23B codon 249, XPG codon 1104, XPC codon 939, XPF codons 415 and 662, and ERCC6 codons 1213 and 1230). Modification of ORs for smoking by single and combined NER genotypes was investigated. In this study population, smoking was more strongly associated with breast cancer in African American women compared with white women. Among African American women, the association of breast cancer and smoking was strongest among women with specific combinations of NER genotypes. Evidence for multiplicative interaction was found between combined NER genotypes and smoking dose (likelihood ratio test P = 0.06), duration (P = 0.09), time since cessation (P = 0.02), age at initiation (P = 0.04) and former smoking (P = 0.03). No interactions were observed in white women. Therefore, polymorphisms in NER genes may modify the relationship between breast cancer and smoking. These results are consistent with previous evidence of exposure-specific p53 mutations in breast tumors from current and former smokers, suggesting that smoking may play a role in breast cancer etiology.

  11. Nucleotide excision repair of 2-acetylaminofluorene- and 2-aminofluorene-(C8)-guanine adducts: molecular dynamics simulations elucidate how lesion structure and base sequence context impact repair efficiencies.

    PubMed

    Mu, Hong; Kropachev, Konstantin; Wang, Lihua; Zhang, Lu; Kolbanovskiy, Alexander; Kolbanovskiy, Marina; Geacintov, Nicholas E; Broyde, Suse

    2012-10-01

    Nucleotide excision repair (NER) efficiencies of DNA lesions can vary by orders of magnitude, for reasons that remain unclear. An example is the pair of N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) adducts that differ by a single acetyl group. The NER efficiencies in human HeLa cell extracts of these lesions are significantly different when placed at G(1), G(2) or G(3) in the duplex sequence (5'-CTCG(1)G(2)CG(3)CCATC-3') containing the NarI mutational hot spot. Furthermore, the dG-C8-AAF adduct is a better substrate of NER than dG-C8-AF in all three NarI sequence contexts. The conformations of each of these adducts were investigated by Molecular dynamics (MD) simulation methods. In the base-displaced conformational family, the greater repair susceptibility of dG-C8-AAF in all sequences stems from steric hindrance effects of the acetyl group which significantly diminish the adduct-base stabilizing van der Waals stacking interactions relative to the dG-C8-AF case. Base sequence context effects for each adduct are caused by differences in helix untwisting and minor groove opening that are derived from the differences in stacking patterns. Overall, the greater NER efficiencies are correlated with greater extents of base sequence-dependent local untwisting and minor groove opening together with weaker stacking interactions.

  12. Three-dimensional single-shot optoacoustic visualization of excised mouse organs with model-based reconstruction

    NASA Astrophysics Data System (ADS)

    Deán-Ben, X. L.; Buehler, Andreas; Ntziachristos, Vasilis; Razansky, Daniel

    2013-03-01

    Optoacoustic imaging offers the unique capability of simultaneous excitation of a three-dimensional (volumetric) region with a single interrogating laser pulse. In this way, three-dimensional imaging with single-shot illumination is theoretically achievable, which in principle allows the visualization of dynamic events at a high frame rate mainly limited by the pulse repetition rate of the laser. Simultaneous acquisition of optoacoustic signals at a set of points surrounding the imaging sample is however required for this purpose, which is hampered by several technical limitations related to lack of appropriate ultrasound detection technology, digital sampling and processing capacities. Also, a convenient reconstruction algorithm must be selected to accurately image the distribution of the optical absorption from the acquired signals. Specifically, the resolution and quantitativeness of the images depend on the reconstruction procedure employed. Herein we describe an accurate three-dimensional model-based optoacoustic reconstruction algorithm based on a convenient discretization of the analytical solution of the forward model. Subsequent algebraic inversion is done with the LSQR algorithm. The performance of the algorithm is showcased by reconstructing an excised mouse heart with a custom made three-dimensional optoacoustic imaging system. In this system, 256 optoacoustic signals corresponding to single-shot excitation are simultaneously collected with an array of ultrasonic transducers disposed on a spherical surface, which allows three-dimensional imaging at a frame rate of 10 Hz.

  13. DNA Based Molecular Scale Nanofabrication

    DTIC Science & Technology

    2015-12-04

    water adsorption on DNA origami template and its impact on DNA-mediated chemical reactions. We also extended the concept of DNA-mediated reaction to...other nanoscale templates, (b) Studied the thermal and chemical stability of DNA origami template. The result shows that the DNA nanostructures can be...potentially used in very harsh chemical environments, (c) Studied the effect of DNA origami template on the growth of self-assembled monolayer (SAM

  14. C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins

    PubMed Central

    Anderson, Edward N; Corkins, Mark E; Li, Jia-Cheng; Singh, Komudi; Parsons, Sadé; Tucey, Tim M; Sorkaç, Altar; Huang, Huiyan; Dimitriadi, Maria; Sinclair, David A

    2016-01-01

    Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5’-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work. PMID:26854551

  15. DNA-based watermarks using the DNA-Crypt algorithm

    PubMed Central

    Heider, Dominik; Barnekow, Angelika

    2007-01-01

    Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

  16. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  17. Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

    PubMed

    Murata-Kamiya, N; Kaji, H; Kasai, H

    1999-06-07

    To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.

  18. BIPHASIC REGULATION OF BASE EXCISION DNA REPAIR BY LOW DOSE ARSENIC. (R826135)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. CRISPR-based screening of genomic island excision events in bacteria.

    PubMed

    Selle, Kurt; Klaenhammer, Todd R; Barrangou, Rodolphe

    2015-06-30

    Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.

  20. Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair.

    PubMed Central

    Wei, D; Maher, V M; McCormick, J J

    1996-01-01

    Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide

  1. Engineering DNA-based functional materials.

    PubMed

    Roh, Young Hoon; Ruiz, Roanna C H; Peng, Songming; Lee, Jong Bum; Luo, Dan

    2011-12-01

    While DNA is a genetic material, it is also an inherently polymeric material made from repeating units called nucleotides. Although DNA's biological functions have been studied for decades, the polymeric features of DNA have not been extensively exploited until recently. In this tutorial review, we focus on two aspects of using DNA as a polymeric material: (1) the engineering methods, and (2) the potential real-world applications. More specifically, various strategies for constructing DNA-based building blocks and materials are introduced based on DNA topologies, which include linear, branched/dendritic, and networked. Different applications in nanotechnology, medicine, and biotechnology are further reviewed.

  2. Method for sequencing DNA base pairs

    DOEpatents

    Sessler, Andrew M.; Dawson, John

    1993-01-01

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source.

  3. DNA base identification by electron microscopy.

    PubMed

    Bell, David C; Thomas, W Kelley; Murtagh, Katelyn M; Dionne, Cheryl A; Graham, Adam C; Anderson, Jobriah E; Glover, William R

    2012-10-01

    Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.

  4. Identification of DNA lesions using a third base pair for amplification and nanopore sequencing.

    PubMed

    Riedl, Jan; Ding, Yun; Fleming, Aaron M; Burrows, Cynthia J

    2015-11-06

    Damage to the genome is implicated in the progression of cancer and stress-induced diseases. DNA lesions exist in low levels, and cannot be amplified by standard PCR because they are frequently strong blocks to polymerases. Here, we describe a method for PCR amplification of lesion-containing DNA in which the site and identity could be marked, copied and sequenced. Critical for this method is installation of either the dNaM or d5SICS nucleotides at the lesion site after processing via the base excision repair process. These marker nucleotides constitute an unnatural base pair, allowing large quantities of marked DNA to be made by PCR amplification. Sanger sequencing confirms the potential for this method to locate lesions by marking, amplifying and sequencing a lesion in the KRAS gene. Detection using the α-hemolysin nanopore is also developed to analyse the markers in individual DNA strands with the potential to identify multiple lesions per strand.

  5. Role of base sequence context in conformational equilibria and nucleotide excision repair of benzo[a]pyrene diol epoxide-adenine adducts.

    PubMed

    Yan, Shixiang; Wu, Min; Buterin, Tonko; Naegeli, Hanspeter; Geacintov, Nicholas E; Broyde, Suse

    2003-03-04

    We investigate the influence of base sequence context on the conformations of the 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts through molecular dynamics (MD) simulations with free energy calculations, and relate the structural findings to results of nucleotide excision repair (NER) assays in human cell extracts. In previous studies, these adducts were studied in the CA*A sequence context, and here we report results for the CA*C sequence. Our simulations indicate that the base sequence context affects the syn-anti conformational equilibrium in the 10S (+) adduct by modulating the barrier heights between these states on the energy surface, with a higher barrier in the CA*C case. Our nucleotide excision repair assay finds greater NER susceptibilities in the 10S (+) adduct for the CA*C sequence context. A structural rationale ties together these results. A sequence specific hydrogen bond, accompanied by a significantly increased roll and consequent bending in the 10S (+) adduct, has been found in our simulations for the CA*C sequence, which could account for the enhanced nucleotide excision repair as well as the syn-anti equilibrium difference we observe in this isomer and sequence. Such sequence specific differential repair could contribute to the existence of mutational hotspots and thereby contribute to the complexity of cancer initiation.

  6. Metallic Nanostructures Based on DNA Nanoshapes

    PubMed Central

    Shen, Boxuan; Tapio, Kosti; Linko, Veikko; Kostiainen, Mauri A.; Toppari, Jari Jussi

    2016-01-01

    Metallic nanostructures have inspired extensive research over several decades, particularly within the field of nanoelectronics and increasingly in plasmonics. Due to the limitations of conventional lithography methods, the development of bottom-up fabricated metallic nanostructures has become more and more in demand. The remarkable development of DNA-based nanostructures has provided many successful methods and realizations for these needs, such as chemical DNA metallization via seeding or ionization, as well as DNA-guided lithography and casting of metallic nanoparticles by DNA molds. These methods offer high resolution, versatility and throughput and could enable the fabrication of arbitrarily-shaped structures with a 10-nm feature size, thus bringing novel applications into view. In this review, we cover the evolution of DNA-based metallic nanostructures, starting from the metallized double-stranded DNA for electronics and progress to sophisticated plasmonic structures based on DNA origami objects. PMID:28335274

  7. Tunable and regenerative DNA zipper based spring

    NASA Astrophysics Data System (ADS)

    Landon, Preston; Mo, Alexander; Ramachandran, Srinivasan; Lal, Ratnesh

    2012-02-01

    We report a DNA zipper based actuator device termed `DNA- spring' with tunable and repeated cycles of extension and contraction ability. DNA zipper is a double-stranded DNA system engineered to open upon its specific interaction with appropriately designed single strand DNA (ssDNA), opening of the zipper is driven by binding energy differences between the DNA strands. The zipper system is incorporated with defined modifications to function like a spring, capable of delivering approximately 9 pN force over a distance of approximately 13 nm, producing approximately 116 kJ/mol of work. Time-lapse fluorescence and fluorescent DNA gel electrophoresis analysis is utilized to evaluate and confirm the spring action. A second zipper incorporated into the spring provides the ability to couple/decouple to an object/substrate. Such devices would have wide application, including for conditionally triggered molecular delivery systems and as actuators in nano-devices. zippers.

  8. Hemangioma excision (image)

    MedlinePlus

    A hemangioma is a non-cancerous (benign) growth of blood vessels. They are the most common benign blood vessel ( ... time and occasionally with medication. Large or disfiguring hemangiomas may require surgical excision.

  9. Reversible Data Hiding Based on DNA Computing

    PubMed Central

    Xie, Yingjie

    2017-01-01

    Biocomputing, especially DNA, computing has got great development. It is widely used in information security. In this paper, a novel algorithm of reversible data hiding based on DNA computing is proposed. Inspired by the algorithm of histogram modification, which is a classical algorithm for reversible data hiding, we combine it with DNA computing to realize this algorithm based on biological technology. Compared with previous results, our experimental results have significantly improved the ER (Embedding Rate). Furthermore, some PSNR (peak signal-to-noise ratios) of test images are also improved. Experimental results show that it is suitable for protecting the copyright of cover image in DNA-based information security. PMID:28280504

  10. Reversible Data Hiding Based on DNA Computing.

    PubMed

    Wang, Bin; Xie, Yingjie; Zhou, Shihua; Zhou, Changjun; Zheng, Xuedong

    2017-01-01

    Biocomputing, especially DNA, computing has got great development. It is widely used in information security. In this paper, a novel algorithm of reversible data hiding based on DNA computing is proposed. Inspired by the algorithm of histogram modification, which is a classical algorithm for reversible data hiding, we combine it with DNA computing to realize this algorithm based on biological technology. Compared with previous results, our experimental results have significantly improved the ER (Embedding Rate). Furthermore, some PSNR (peak signal-to-noise ratios) of test images are also improved. Experimental results show that it is suitable for protecting the copyright of cover image in DNA-based information security.

  11. Method for sequencing DNA base pairs

    DOEpatents

    Sessler, A.M.; Dawson, J.

    1993-12-14

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source. 6 figures.

  12. DNA Based Electrochromic and Photovoltaic Cells

    DTIC Science & Technology

    2012-01-01

    SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON a. REPORT...9550-09-1-0647 final 01-09-2009 ; 30-11-2011 DNA Based Electrochromic and Photovoltaic Cells FA 9550-09-1-0647 Pawlicka, Agnieszka, J. Instituto de...as well as DNA-CTMA and DNA-DODA were also obtained and characterized. High ionic conductivity results combined with transparency and adhesion to the

  13. Nucleotide excision repair in Escherichia coli.

    PubMed Central

    Van Houten, B

    1990-01-01

    One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation. Escherichia coli has served as a model organism for the study of this process. Recently, many of the proteins that mediate E. coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway. One of the key repair enzymes of this pathway is the UvrABC nuclease complex. The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide. The UvrABC complex displays a remarkable substrate diversity. Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction. Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex. Current research is devoted to understanding how these complex events are mediated within the living cell. PMID:2181258

  14. Alternative DNA base pairing through metal coordination.

    PubMed

    Clever, Guido H; Shionoya, Mitsuhiko

    2012-01-01

    Base-pairing in the naturally occurring DNA and RNA oligonucleotide duplexes is based on π-stacking, hydrogen bonding, and shape complementarity between the nucleobases adenine, thymine, guanine, and cytosine as well as on the hydrophobic-hydrophilic balance in aqueous media. This complex system of multiple supramolecular interactions is the product of a long-term evolutionary process and thus highly optimized to serve its biological functions such as information storage and processing. After the successful implementation of automated DNA synthesis, chemists have begun to introduce artificial modifications inside the core of the DNA double helix in order to study various aspects of base pairing, generate new base pairs orthogonal to the natural ones, and equip the biopolymer with entirely new functions. The idea to replace the hydrogen bonding interactions with metal coordination between ligand-like nucleosides and suitable transition metal ions culminated in the development of a plethora of artificial base-pairing systems termed "metal base-pairs" which were shown to strongly enhance the DNA duplex stability. Furthermore, they show great potential for the use of DNA as a molecular wire in nanoscale electronic architectures. Although single electrons have proven to be transmitted by natural DNA over a distance of several base pairs, the high ohmic resistance of unmodified oligonucleotides was identified as a serious obstacle. By exchanging some or all of the Watson-Crick base pairs in DNA with metal complexes, this problem may be solved. In the future, these research efforts are supposed to lead to DNA-like materials with superior conductivity for nano-electronic applications. Other fields of potential application such as DNA-based supramolecular architecture and catalysis may be strongly influenced by these developments as well. This text is meant to illustrate the basic concepts of metal-base pairing and give an outline over recent developments in this field.

  15. QPSO-based adaptive DNA computing algorithm.

    PubMed

    Karakose, Mehmet; Cigdem, Ugur

    2013-01-01

    DNA (deoxyribonucleic acid) computing that is a new computation model based on DNA molecules for information storage has been increasingly used for optimization and data analysis in recent years. However, DNA computing algorithm has some limitations in terms of convergence speed, adaptability, and effectiveness. In this paper, a new approach for improvement of DNA computing is proposed. This new approach aims to perform DNA computing algorithm with adaptive parameters towards the desired goal using quantum-behaved particle swarm optimization (QPSO). Some contributions provided by the proposed QPSO based on adaptive DNA computing algorithm are as follows: (1) parameters of population size, crossover rate, maximum number of operations, enzyme and virus mutation rate, and fitness function of DNA computing algorithm are simultaneously tuned for adaptive process, (2) adaptive algorithm is performed using QPSO algorithm for goal-driven progress, faster operation, and flexibility in data, and (3) numerical realization of DNA computing algorithm with proposed approach is implemented in system identification. Two experiments with different systems were carried out to evaluate the performance of the proposed approach with comparative results. Experimental results obtained with Matlab and FPGA demonstrate ability to provide effective optimization, considerable convergence speed, and high accuracy according to DNA computing algorithm.

  16. Excision and duplication of su3+-transducing fragments carried by bacteriophage phi 80. I. Novel structure of phi 80sus2psu3+ DNA molecule.

    PubMed Central

    Yamagishi, H; Inokuchi, H; Ozeki, H

    1976-01-01

    DNA molecules of phi 80sus2psu3+ and phi 80dsu3+ isolated by Andoh and Ozeki (1968) were studied by the electron microscope heteroduplex method. The phi 80sus2psu3+ and phi 80dsu3+ DNA lengths were found to be 108.7 and 103.3% of the phi 80 DNA, respectively. The phi 80sus2psu3+/phi 80 heteroduplex shows an insertion loop of 8.7% of the phi 80 DNA which migrates from 7.7 to 9.7%, as measured relative to the left (0%) and right (100%) termini of the mature phi 80 DNA molecule. The region of loop migration occupies the central region of the phi 80 head gene cluster. The presence of su3+-containing Escherichia coli DNA of 6.7% phi 80 unit flanked by two homologous regions of phage DNA of 2.0% of phi 80 unit gives rise to a movable insertion loop. In phi 80dsu3+, from which phi 80sus2psu3+ was derived, 50.5% of the phi 80 DNA at the left arm was replaced by E. coli DNA containing the su3+ gene, equivalent to about 53.8% phi 80 unit in length. The phi 80sus2psu3+/phi 80dsu3+ heteroduplex appears as a double-stranded molecule that bifurcates into two clearly visible single-stranded regions, rejoins, bifurcates, and rejoins again. The middle double-stranded stretches of 6.7% phi 80 unit correspond to the E. coli DNA inserted in phi 80sus2psu3+. Therefore the transducing fragment carried by phi 80sus2psu3+ originates from the inside region of the transducing fragment of defective phage phi 80dsu3+ by at least two illegitimate recombination events. Images PMID:1271527

  17. Single-quantum-dot-based DNA nanosensor

    NASA Astrophysics Data System (ADS)

    Zhang, Chun-Yang; Yeh, Hsin-Chih; Kuroki, Marcos T.; Wang, Tza-Huei

    2005-11-01

    Rapid and highly sensitive detection of DNA is critical in diagnosing genetic diseases. Conventional approaches often rely on cumbersome, semi-quantitative amplification of target DNA to improve detection sensitivity. In addition, most DNA detection systems (microarrays, for example), regardless of their need for target amplification, require separation of unhybridized DNA strands from hybridized stands immobilized on a solid substrate, and are thereby complicated by solution-surface binding kinetics. Here, we report an ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET) capable of detecting low concentrations of DNA in a separation-free format. This system uses quantum dots (QDs) linked to DNA probes to capture DNA targets. The target strand binds to a dye-labelled reporter strand thus forming a FRET donor-acceptor ensemble. The QD also functions as a concentrator that amplifies the target signal by confining several targets in a nanoscale domain. Unbound nanosensors produce near-zero background fluorescence, but on binding to even a small amount of target DNA (~50 copies or less) they generate a very distinct FRET signal. A nanosensor-based oligonucleotide ligation assay has been demonstrated to successfully detect a point mutation typical of some ovarian tumours in clinical samples.

  18. Crosstalk between MSH2–MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair

    PubMed Central

    Lai, Yanhao; Budworth, Helen; Beaver, Jill M.; Chan, Nelson L. S.; Zhang, Zunzhen; McMurray, Cynthia T.; Liu, Yuan

    2016-01-01

    Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion. PMID:27546332

  19. DNA-Based Enzyme Reactors and Systems

    PubMed Central

    Linko, Veikko; Nummelin, Sami; Aarnos, Laura; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.

    2016-01-01

    During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications. PMID:28335267

  20. A scoring system basing pathological parameters to predict regional lymph node metastasis after preoperative chemoradiotherapy for locally advanced rectal cancer: implication for local excision

    PubMed Central

    Wang, Xiao-Jie; Chi, Pan; Lin, Hui-Ming; Lu, Xing-Rong; Huang, Ying; Xu, Zong-Bin; Huang, Sheng-Hui; Sun, Yan-Wu; Ye, Dao-Xiong; Yu, Qian

    2016-01-01

    Local excision is an alternative to radical surgery that is indicated in patients with locally advanced rectal cancer (LARC) who have a good response to chemoradiotherapy (CRT). Regional lymph node status is a major uncertainty during local excision of LARC following CRT. We retrospectively reviewed clinicopathologic variables for 244 patients with LARC who were treated at our institute between December 2000 and December 2013 in order to identify independent predictors of regional lymph node metastasis. Multivariate analysis of the training sample demonstrated that histopathologic type, tumor size, and the presence of lymphovascular invasion were significant predictors of regional nodal metastasis. These variables were then incorporated into a scoring system in which the total scores were calculated based on the points assigned for each parameter. The area under the curve in the receiver operating characteristic analysis was 0.750, and the cutoff value for the total score to predict regional nodal metastasis was 7.5. The sensitivity of our system was 73.2% and the specificity was 69.4%. The sensitivity was 77.8% and the specificity was 51.2% when the scoring system was applied to the testing sample. Using this system, we could accurately predict regional nodal metastases in LARC patients following CRT, which may be useful for stratifying patients in clinical trials and selecting potential candidates for organ-sparing surgery following CRT for LARC PMID:27489356

  1. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    SciTech Connect

    Gupta, P.K.; Sirover, M.A.

    1984-10-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. 62 references, 3 figures, 2 tables.

  2. Laparoscopic Total Mesorectum Excision

    PubMed Central

    Quilici, F.A.; Cordeiro, F.; Reis, J.A.; Kagohara, O.; Simões Neto, J.

    2002-01-01

    The main controversy of colon-rectal laparoscopic surgery comes from its use as a cancer treatment. Two points deserve special attention: the incidence of portsite tumor implantation and the possibility of performing radical cancer surgery, such as total mesorectum excision. Once these points are addressed, the laparoscopic approach will be used routinely to treat rectal cancer. To clarify these points, 32 patients with cancer of the lower rectum participated in a special protocol that included preoperative radiotherapy and laparoscopic total mesorectum excision. All data were recorded. At the same time, all data recorded from the experience of a multicenter laparoscopic group (Brazilian Colorectal Laparoscopic Surgeons – 130 patients with tumor of the lower rectum) were analyzed and compared with the data provided by our patients. Analysis of the results suggests that a laparoscopic approach allows the same effective resection as that of conventional surgery and that preoperative irradiation does not influence the incidence of intraoperative complications. The extent of lymph nodal excision is similar to that obtained with open surgery, with an average of 12.3 lymph nodes dissected per specimen. The rate of local recurrence was 3.12%. No port site implantation of tumor was noted in this series of patients with cancer of the lower rectum. PMID:12113422

  3. Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair

    PubMed Central

    Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Wani, Altaf

    2012-01-01

    Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in Xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER. PMID:23083890

  4. Unzipping of DNA with correlated base sequence.

    PubMed

    Allahverdyan, A E; Gevorkian, Zh S; Hu, Chin-Kun; Wu, Ming-Chya

    2004-06-01

    We consider force-induced unzipping transition for a heterogeneous DNA model with a correlated base sequence. Both finite-range and long-range correlated situations are considered. It is shown that finite-range correlations increase stability of DNA with respect to the external unzipping force. Due to long-range correlations the number of unzipped base pairs displays two widely different scenarios depending on the details of the base sequence: either there is no unzipping phase transition at all, or the transition is realized via a sequence of jumps with magnitude comparable to the size of the system. Both scenarios are different from the behavior of the average number of unzipped base pairs (non-self-averaging). The results can be relevant for explaining the biological purpose of correlated structures in DNA.

  5. Arthroscopic excision of ganglion cysts.

    PubMed

    Bontempo, Nicholas A; Weiss, Arnold-Peter C

    2014-02-01

    Arthroscopy is an advancing field in orthopedics, the applications of which have been expanding over time. Traditionally, excision of ganglion cysts has been done in an open fashion. However, more recently, studies show outcomes following arthroscopic excision to be as good as open excision. Cosmetically, the incisions are smaller and heal faster following arthroscopy. In addition, there is the suggested benefit that patients will regain function and return to work faster following arthroscopic excision. More prospective studies comparing open and arthroscopic excision of ganglion cysts need to be done in order to delineate if there is a true functional benefit.

  6. Power of surface-based DNA computation

    SciTech Connect

    Cai, Weiping; Condon, A.E.; Corn, R.M.

    1997-12-01

    A new model of DNA computation that is based on surface chemistry is studied. Such computations involve the manipulation of DNA strands that are immobilized on a surface, rather than in solution as in the work of Adleman. Surface-based chemistry has been a critical technology in many recent advances in biochemistry and offers several advantages over solution-based chemistry, including simplified handling of samples and elimination of loss of strands, which reduce error in the computation. The main contribution of this paper is in showing that in principle, surface-based DNA chemistry can efficiently support general circuit computation on many inputs in parallel. To do this, an abstract model of computation that allows parallel manipulation of binary inputs is described. It is then shown that this model can be implemented by encoding inputs as DNA strands and repeatedly modifying the strands in parallel on a surface, using the chemical processes of hybridization, exonuclease degradation, polymerase extension, and ligation. Thirdly, it is shown that the model supports efficient circuit simulation in the following sense: exactly those inputs that satisfy a circuit can be isolated and the number of parallel operations needed to do this is proportional to the size of the circuit. Finally, results are presented on the power of the model when another resource of DNA computation is limited, namely strand length. 12 refs.

  7. Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

    PubMed

    Ma, Huaxian; Wang, Jianling; Abdel-Rahman, Sherif Z; Boor, Paul J; Khan, M Firoze

    2013-03-15

    Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of

  8. Multiphoton excitation of fluorescent DNA base analogs.

    PubMed

    Katilius, Evaldas; Woodbury, Neal W

    2006-01-01

    Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5 x 10(-50) cm(4)s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

  9. Mechanism-based drug combinations with the DNA-strand-breaking nucleoside analog, CNDAC

    PubMed Central

    Liu, Xiaojun; Jiang, Yingjun; Nowak, Billie; Hargis, Sarah; Plunkett, William

    2016-01-01

    CNDAC (2’-C-cyano-2’-deoxy-1-β-D-arabino-pentofuranosyl-cytosine, DFP10917) and its orally bioavailable prodrug, sapacitabine, are undergoing clinical trials for hematological malignancies and solid tumors. The unique action mechanism of inducing DNA strand breaks distinguishes CNDAC from other deoxycytidine analogs. To optimize the clinical potentials of CNDAC, we explored multiple strategies combining CNDAC with chemotherapeutic agents targeting distinct DNA damage repair pathways that are currently in clinical use. The ability of each agent to decrease proliferative potential, determined by clonogenic assays, was determined in paired cell lines proficient and deficient in certain DNA repair proteins. Subsequently each agent was used in combination with CNDAC at fixed concentration ratios. The clonogenicity was quantitated by median effect analysis, and a combination index was calculated. The c-Abl kinase inhibitor, imatinib, had synergy with CNDAC in HCT116 cells, regardless of p53 status. Inhibitors of PARP1 that interfere with homologous recombination (HR) repair or base excision repair (BER) and agents such as temozolomide that cause DNA damage repaired by the BER pathway were also synergistic with CNDAC. The toxicity of the nitrogen mustards, bendamustine and cytoxan, or of platinum compounds, which generate DNA adducts repaired by nucleotide excision repair and HR, was additive with CNDAC. An additive cell killing was also achieved by the combination of CNDAC with taxane mitotic inhibitors (paclitaxel and docetaxel). At concentrations which allow survival of the majority of wild type cells, the synergistic or additive combination effects were selective in HR-deficient cells. This study provides mechanistic rationales for combining CNDAC with other active drugs. PMID:27474148

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

    PubMed

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

    2015-01-01

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

  11. A Base-Independent Repair Mechanism for DNA Glycosylase—No Discrimination Within the Active Site

    PubMed Central

    Blank, Iris D.; Sadeghian, Keyarash; Ochsenfeld, Christian

    2015-01-01

    The ubiquitous occurrence of DNA damages renders its repair machinery a crucial requirement for the genomic stability and the survival of living organisms. Deficiencies in DNA repair can lead to carcinogenesis, Alzheimer, or Diabetes II, where increased amounts of oxidized DNA bases have been found in patients. Despite the highest mutation frequency among oxidized DNA bases, the base-excision repair process of oxidized and ring-opened guanine, FapydG (2,6-diamino-4-hydroxy-5-formamidopyrimidine), remained unclear since it is difficult to study experimentally. We use newly-developed linear-scaling quantum-chemical methods (QM) allowing us to include up to 700 QM-atoms and achieving size convergence. Instead of the widely assumed base-protonated pathway we find a ribose-protonated repair mechanism which explains experimental observations and shows strong evidence for a base-independent repair process. Our results also imply that discrimination must occur during recognition, prior to the binding within the active site. PMID:26013033

  12. Random Coding Bounds for DNA Codes Based on Fibonacci Ensembles of DNA Sequences

    DTIC Science & Technology

    2008-07-01

    COVERED (From - To) 6 Jul 08 – 11 Jul 08 4. TITLE AND SUBTITLE RANDOM CODING BOUNDS FOR DNA CODES BASED ON FIBONACCI ENSEMBLES OF DNA SEQUENCES ... sequences which are generalizations of the Fibonacci sequences . 15. SUBJECT TERMS DNA Codes, Fibonacci Ensembles, DNA Computing, Code Optimization 16...coding bound on the rate of DNA codes is proved. To obtain the bound, we use some ensembles of DNA sequences which are generalizations of the Fibonacci

  13. RFI Mitigation / Excision techniques

    NASA Astrophysics Data System (ADS)

    Roshi, D. A.

    2004-06-01

    Radio frequency interference (RFI) is increasingly affecting radio astronomy research. A few years ago, active research to investigate the possibility of observing in the presence of interference using RFI mitigation techniques was initiated. In this paper, I briefly discuss four RFI mitigation/excision projects. These projects are:- (1) A technique to suppress double sideband amplitude modulated interference in which I show that an astronomical signal in the presence of a DSB interference can be observed with a signal-to-noise ratio factor of 2 less compared to observations if the RFI were not present. (2) Techniques to suppress interference due to synchronization signals in composite video signals are presented. A combination of noise-free modelling of the synchronization signals and adaptive filtering is used for suppressing the interference. (3) Design techniques to minimize spurious pick-up at the analog input of an analog-to-digital converter are discussed. (4) Spectral RFI excision using a spectral channel weighted scheme and its application to Green Bank telescope observations are also presented.

  14. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  15. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  16. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks

    PubMed Central

    Wyatt, Michael D.; Pittman, Douglas L.

    2008-01-01

    The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a ‘radiomimetic,’ i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired. PMID:17173371

  17. Biosensors based on DNA-Functionalized Graphene

    NASA Astrophysics Data System (ADS)

    Vishnubhotla, Ramya; Ping, Jinglei; Vrudhula, Amey; Johnson, A. T. Charlie

    Since its discovery, graphene has been used for sensing applications due to its outstanding electrical properties and biocompatibility. Here, we demonstrate the capabilities of field effect transistors (FETs) based on CVD-grown graphene functionalized with commercially obtained DNA oligomers and aptamers for detection of various biomolecular targets (e.g., complementary DNA and small molecule drug targets). Graphene FETs were created with a scalable photolithography process that produces arrays consisting of 50-100 FETs with a layout suitable for multiplexed detection of four molecular targets. FETs were characterized via AFM to confirm the presence of the aptamer. From the measured electrical characteristics, it was determined that binding of molecular targets by the DNA chemical recognition element led to a reproducible, concentration-dependent shift in the Dirac voltage. This biosensor class is potentially suitable for applications in drug detection. This work is funded by NIH through the Center for AIDS Research at the University of Pennsylvania.

  18. DNA-Based Nanostructures: Changes of Mechanical Properties of DNA upon Ligand Binding

    NASA Astrophysics Data System (ADS)

    Nechipurenko, Yury; Grokhovsky, Sergey; Gursky, Georgy; Nechipurenko, Dmitry; Polozov, Robert

    The formation of DNA-based nanostructures involves the binding of different kinds of ligands to DNA as well as the interaction of DNA molecules with each other. Complex formation between ligand and DNA can alter physicochemical properties of the DNA molecule. In the present work, the accessibility of DNA-ligand complexes to cleavage by DNase I are considered, and the exact algorithms for analysis of diagrams of DNase I footprinting for ligand-DNA complexes are obtained. Changes of mechanical properties of the DNA upon ligand binding are also demonstrated by the cleavage patterns generated upon ultrasound irradiation of cis-platin-DNA complexes. Propagation of the mechanical perturbations along DNA in the presence of bound ligands is considered in terms of a string model with a heterogeneity corresponding to the position of a bound ligand on DNA. This model can reproduce qualitatively the cleavage patterns obtained upon ultrasound irradiation of cis-platin-DNA complexes.

  19. [Ionizing radiation-induced DNA damage and its repair in human cells]. Progress report, [April 1, 1993--February 28, 1994

    SciTech Connect

    Not Available

    1994-07-01

    The excision of radiation-induced lesions in DNA by a DNA repair enzyme complex, namely the UvrABC nuclease complex, has been investigated. Irradiated DNA was treated with the enzyme complex. DNA fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results showed that a number pyrimidine- and purine-derived lesions in DNA were excised by the UvrABC nuclease complex and that the enzyme complex does not act on radiation-induced DNA lesions as a glycosylase. This means that it does not excise individual base products, but it excises oligomers containing these lesions. A number of pyrimidine-derived lesions that were no substrates for other DNA repair enzymes investigated in our laboratory were substrates for the UvrABC nuclease complex.

  20. Communication: Electron ionization of DNA bases

    NASA Astrophysics Data System (ADS)

    Rahman, M. A.; Krishnakumar, E.

    2016-04-01

    No reliable experimental data exist for the partial and total electron ionization cross sections for DNA bases, which are very crucial for modeling radiation damage in genetic material of living cell. We have measured a complete set of absolute partial electron ionization cross sections up to 500 eV for DNA bases for the first time by using the relative flow technique. These partial cross sections are summed to obtain total ion cross sections for all the four bases and are compared with the existing theoretical calculations and the only set of measured absolute cross sections. Our measurements clearly resolve the existing discrepancy between the theoretical and experimental results, thereby providing for the first time reliable numbers for partial and total ion cross sections for these molecules. The results on fragmentation analysis of adenine supports the theory of its formation in space.

  1. DNA methylation profiling using bisulfite-based epityping of pooled genomic DNA.

    PubMed

    Docherty, Sophia J; Davis, Oliver S P; Haworth, Claire M A; Plomin, Robert; Mill, Jonathan

    2010-11-01

    DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however they require large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. DNA pooling approaches are already widely used in large-scale studies of DNA sequence and gene expression. In this paper, we describe the application of this economical DNA pooling technique to the study of DNA methylation profiles. This method generates accurate quantitative assessments of group DNA methylation averages, reducing the time, cost and amount of DNA starting material required for large-scale epigenetic investigation of disease phenotypes.

  2. [Mesocolic excision for colonic adenocarcinoma].

    PubMed

    Debove, Clotilde; Lefèvre, Jérémie H; Parc, Yann

    2017-02-01

    On the same principle than total mesorectal excision in rectal cancer, the effect of complete mesocolic excision on short and long-term outcomes is actually evaluated for colonic adenocarcinoma. This method, usually performed for left colectomy, offers a surgical specimen of higher quality, with a larger number of lymph nodes harvested. For right colectomy, surgical specifications make it less common complete mesocolic excision and conventional surgery offer comparable outcomes, as regards to postoperative morbidity and mortality rates. No differences are identified between laparoscopic and open surgery. On oncologic outcomes, only two studies report a higher free-disease survival after complete mesocolic excision. Then, there is evidence that complete mesocolic excision offers a higher rate of specimen with extensive lymph node resection, without increased morbidity rate. However, there is limited evidence that it leads to improve long-term oncological outcomes.

  3. Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts.

    PubMed

    You, Changjun; Wang, Yinsheng

    2016-02-16

    The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) in cells. We also found that the N(2)-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription

  4. Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA.

    PubMed

    Yokoya, A; Fujii, K; Ushigome, T; Shikazono, N; Urushibara, A; Watanabe, R

    2006-01-01

    The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH*, with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect.

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

    PubMed

    Rhee, David B; Ghosh, Avik; Lu, Jian; Bohr, Vilhelm A; Liu, Yie

    2011-01-02

    Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non-telomeric double-stranded substrates. In addition, telomere repeat binding factors TRF1 and TRF2 do not impair OGG1 incision activity. Yet, 8-oxodG in some telomere structures (e.g., fork-opening, 3'-overhang, and D-loop) were less effectively excised by OGG1, depending upon its position in these substrates. Collectively, our data indicate that the sequence context of telomere repeats and certain telomere configurations may contribute to telomere vulnerability to oxidative DNA damage processing.

  6. DNA sequencing by synthesis based on elongation delay detection

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2015-03-01

    The one of most important problem in modern genetics, biology and medicine is determination of the primary nucleotide sequence of the DNA of living organisms (DNA sequencing). This paper describes the label-free DNA sequencing approach, based on the observation of a discrete dynamics of DNA sequence elongation phase. The proposed DNA sequencing principle are studied by numerical simulation. The numerical model for proposed label-free DNA sequencing approach is based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) and dynamics of nucleotides incorporation to rising DNA strand. The estimates for number of copied DNA sequences for required probability of nucleotide incorporation event detection and correct DNA sequence determination was obtained. The proposed approach can be applied at all known DNA sequencing devices with "sequencing by synthesis" principle of operation.

  7. Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: excision of dimers in cell extracts

    SciTech Connect

    Reynolds, R.J.; Love, J.D.; Friedberg, E.C.

    1981-08-01

    Cell-free extracts prepared from rad1-19, rad2-2, rad3-1, rad4-3, rad7-1, rad10-1, rad14-1, rad16-1, and cycl-1 (rad7) mutants of Saccharomyces cerevisiae all catalyze the preferential excision of thymine-containing pyrimidine dimers from ultraviolet-irradiated DNA specifically incised with M. luteus ultraviolet deoxyribonucleic acid incising activity.

  8. Noninvasive measurement of aristolochic acid-DNA adducts in urine samples from aristolochic acid-treated rats by liquid chromatography coupled tandem mass spectrometry: evidence for DNA repair by nucleotide-excision repair mechanisms.

    PubMed

    Leung, Elvis M K; Chan, Wan

    2014-01-01

    Nephrotoxic aristolochic acids (AAs) form covalently bonded DNA adducts upon metabolic activation. In this work, a non-invasive approach to detect AAs exposure by quantifying urinary excreted DNA-AA adducts is presented. The developed method entails solid-phase extraction (SPE) enrichment of the urine-excreted DNA-AAs adducts, addition of internal standard, and quantification by liquid chromatography coupled tandem mass spectrometric (LC-MS/MS) analysis. Quantitative analysis revealed 7-(deoxyadenosine-N(6)-yl)-aristolactam II and 7-(deoxyguanosine-N(2)-yl)-aristolactam I that were previously detected as major DNA-AA adducts in different organs of AA-dosed rats, were detected as the major urine excreted adducts. Lower levels of 7-(deoxyadenosine-N(6)-yl)-aristolactam I and 7-(deoxyguanosine-N(2)-yl)-aristolactam II were also detected in the collected urine samples. The identities of the detected urinary DNA-AA adducts were confirmed by comparing chromatographic retention time with synthetic standards, by high-accuracy MS, and MS/MS analyses. LC-MS/MS analysis of the urine samples collected from the AAs-dosed rats demonstrated a time-dependent decrease in the urinary adduct levels, indicating the urinary DNA-AA adduct levels were reflective of the tissue adduct levels. It is expected that the developed approach of detecting urinary DNA-AA adducts will facilitate further carcinogenesis investigations of AAs.

  9. Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

    SciTech Connect

    Guliaev, Anton B.; Singer, B.; Hang, Bo

    2004-05-05

    Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA than that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study.

  10. Excess Electron Localization in Solvated DNA Bases

    SciTech Connect

    Smyth, Maeve; Kohanoff, Jorge

    2011-06-10

    We present a first-principles molecular dynamics study of an excess electron in condensed phase models of solvated DNA bases. Calculations on increasingly large microsolvated clusters taken from liquid phase simulations show that adiabatic electron affinities increase systematically upon solvation, as for optimized gas-phase geometries. Dynamical simulations after vertical attachment indicate that the excess electron, which is initially found delocalized, localizes around the nucleobases within a 15 fs time scale. This transition requires small rearrangements in the geometry of the bases.

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  12. New synthetic substrates of mammalian nucleotide excision repair system

    PubMed Central

    Evdokimov, Alexey; Petruseva, Irina; Tsidulko, Aleksandra; Koroleva, Ludmila; Serpokrylova, Inna; Silnikov, Vladimir; Lavrik, Olga

    2013-01-01

    DNA probes for the studies of damaged strand excision during the nucleotide excision repair (NER) have been designed using the novel non-nucleosidic phosphoramidite reagents that contain N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nFlu) moieties. New lesion-imitating adducts being inserted into DNA show good substrate properties in NER process. Modified extended linear nFlu– and nAntr–DNA are suitable for estimation of specific excision activity catalysed with mammalian whole-cell extracts. The following substrate activity range was revealed for the model 137-bp linear double-stranded DNA: nAnt–DNA ≈ nFlu–DNA > Chol–DNA (Chol–DNA—legitimate NER substrate that contains non-nucleoside fragment bearing cholesterol residue). In vitro assay shows that modified DNA can be a useful tool to study NER activity in whole-cell extracts. The developed approach should be of general use for the incorporation of NER-sensitive distortions into model DNAs. The new synthetic extended linear DNA containing bulky non-nucleoside modifications will be useful for NER mechanism study and for applications. PMID:23609543

  13. A simple DNA-based translation system.

    PubMed

    Garibotti, Alejandra V; Liao, Shiping; Seeman, Nadrian C

    2007-02-01

    We have used DNA double crossover (DX) molecules to produce a translation system that generates unique molecular products. The particular species of DX molecule used contains an even number of half-turns between crossover points, so there is a continuous strand on both sides of the molecule. One of these strands acts as the input strand containing the message, and a second strand acts as the product of translation. The crossover strands carry the "code" that connects the two sides of the molecule. This system is more robust, more extendable, and simpler than previous DNA-based translation systems that have been reported. It is designed to be useful in a variety of applications that utilize the concept of translating from one code to another.

  14. Towards DNA-Based Programmable Matter

    DTIC Science & Technology

    2012-02-28

    that moves along a DNA track and mediates autonomous multistep organic synthesis in a single isothermal solution.35 McKee et al. recently used a DNA...functionalize a variety of mesoscale materials with DNA oligonucleotides. Finally, we demonstrated the ability of these DNA-linked surfaces to mediate ...these DNA-linked surfaces to mediate surface-surface interactions in a DNA sequence-specific manner. In a second line of research, we applied these

  15. Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer

    SciTech Connect

    Carles, Joan . E-mail: jcarles@imas.imim.es; Monzo, Mariano; Amat, Marta; Jansa, Sonia; Artells, Rosa; Navarro, Alfons; Foro, Palmira; Alameda, Francesc; Gayete, Angel; Gel, Bernat; Miguel, Maribel; Albanell, Joan; Fabregat, Xavier

    2006-11-15

    Purpose: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. Methods and Materials: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. Results: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p 0.0002). Conclusions: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC.

  16. Radiation-induced degradation of DNA bases

    NASA Astrophysics Data System (ADS)

    Douki, T.; Delatour, T.; Martini, R.; Cadet, J.

    1999-01-01

    Radio-induced degradation of DNA involves radical processes. A series of lesions among the major bases degradation products has been measured in isolated DNA exposed to gamma radiation in aerated aqueous solution. Degradation can be accounted for by the formation of hydroxyl radicals upon radiolysis of water (indirect effect). The four bases are degraded in high yield. Direct effect has been mimicked by photo-induced electron abstraction from the bases producing their radical cation. Quantification of the modified bases showed that guanine is the preferential target. This can be explained by its lower oxidation potential and charge transfer phenomena. La décomposition radio-induite de l'ADN fait intervenir des processus radicalaires. Une série de lésions choisies parmi les produits majeurs de dégradation des bases a été mesurée dans de l'ADN isolé exposé au rayonnement en solution aqueuse aérée. Les modifications sont alors dues aux radicaux hydroxyles produits par la radiolyse de l'eau (effet indirect) et les quatre bases sont efficacement dégradées. L'arrachement d'électrons aux bases par photosensibilisation pour produire leur radical cation, a été utilisé comme modèle de l'effet direct. La quantification des bases modifiées montre que la guanine est préférentiellement dégradée. Cette observation peut s'expliquer par le plus faible potentiel d'oxydation de cette base ainsi que par les phénomènes de transfert de charge vers les guanines.

  17. DNA biosensors based on self-assembled carbon nanotubes.

    PubMed

    Wang, S G; Wang, Ruili; Sellin, P J; Zhang, Qing

    2004-12-24

    DNA biosensors based on self-assembled multi-walled carbon nanotubes (MWNTs) were described in this paper, in which the probe DNA oligonucleotides were immobilized by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA oligonucleotides. Hybridization between the probe and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of the indicator of methylene blue. Our results demonstrate that the DNA biosensors based on self-assembled MWNTs had a higher hybridization efficiency compared to those based on random MWNTs. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.

  18. Rational Inhibitors of DNA Base Excision Repair Enzymes: New Tools for Elucidating the Role of BER in Cancer Chemotherapy

    DTIC Science & Technology

    2006-05-01

    Figure 6A). However, a secondary plot of the Lineweaver - Burk slopes against [3-(3)-27] showed a parabolic response consistent with the presence of at...uncompetitive mechanism (Scheme 1). Like its 6-substituted analogue, initial inspection of the Lineweaver - Burk analysis of 2-(2)-13 indicates mixed-type...the Lineweaver - Burk slopes versus 2-(2)-13 concentration is hyperbolic, indicating that binding of 2-(2)-13 results in partial inhibition (Figure 6B

  19. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    PubMed Central

    1984-01-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation. PMID:6480691

  20. Alkaline unwinding flow cytometry assay to measure nucleotide excision repair.

    PubMed

    Thyagarajan, Bharat; Anderson, Kristin E; Lessard, Christopher J; Veltri, Gregory; Jacobs, David R; Folsom, Aaron R; Gross, Myron D

    2007-03-01

    Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

  1. Structural basis of HIV-1 resistance to AZT by excision

    SciTech Connect

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D.; Clark, Jr., Arthur D.; Hou, Xiaorong; Frenkel, Yulia V.; Gaffney, Barbara L.; Jones, Roger A.; Boyer, Paul L.; Hughes, Stephen H.; Sarafianos, Stefan G.; Arnold, Eddy

    2011-11-23

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  2. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  3. Substrate specificity and sequence-dependent activity of the Saccharomyces cerevisiae 3-methyladenine DNA glycosylase (Mag).

    PubMed

    Lingaraju, Gondichatnahalli M; Kartalou, Maria; Meira, Lisiane B; Samson, Leona D

    2008-06-01

    DNA glycosylases initiate base excision repair by first binding, then excising aberrant DNA bases. Saccharomyces cerevisiae encodes a 3-methyladenine (3MeA) DNA glycosylase, Mag, that recognizes 3MeA and various other DNA lesions including 1,N6-ethenoadenine (epsilon A), hypoxanthine (Hx) and abasic (AP) sites. In the present study, we explore the relative substrate specificity of Mag for these lesions and in addition, show that Mag also recognizes cisplatin cross-linked adducts, but does not catalyze their excision. Through competition binding and activity studies, we show that in the context of a random DNA sequence Mag binds epsilon A and AP-sites the most tightly, followed by the cross-linked 1,2-d(ApG) cisplatin adduct. While epsilon A binding and excision by Mag was robust in this sequence context, binding and excision of Hx was extremely poor. We further studied the recognition of epsilon A and Hx by Mag, when these lesions are present at different positions within A:T and G:C tracts. Overall, epsilon A was slightly less well excised from each position within the A:T and G:C tracts compared to excision from the random sequence, whereas Hx excision was greatly increased in these sequence contexts (by up to 7-fold) compared to the random sequence. However, given most sequence contexts, Mag had a clear preference for epsilon A relative to Hx, except in the TTXTT (X=epsilon A or Hx) sequence context from which Mag removed both lesions with almost equal efficiency. We discuss how DNA sequence context affects base excision by various 3MeA DNA glycosylases.

  4. Strandwise translocation of a DNA glycosylase on undamaged DNA

    SciTech Connect

    Qi, Yan; Nam, Kwangho; Spong, Marie C.; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L.

    2012-05-14

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

  5. [Effect of estrogen on nucleotide excision repair of N2a neuroblastoma cells].

    PubMed

    Yoshioka, Akira; Yamamoto, Aya; Mori, Toshio; Nakamura, Yu; Morikawa, Masayuki; Yoshino, Hiroki; Kiuchi, Kuniaki; Makinodan, Manabu; Kishimoto, Toshifumi

    2007-04-01

    Until now reduced estrogen level has been considered to affect some psychiatric symptoms, because there are sex differences in onset of Schizophrenia and Alzheimer's disease. Estrogen is associated with cognitive functions, and it has been reported to protect oxidative damage of DNA related to base excision repair (BER). Some patients with Xeroderma Pigmentosum, who have normal BER and impaired nucleotide excision repair (NER), are known to be suffering from mental retardation. Therefore we hypothesized that impaired NER was partly associated with pathology of mental disorder and investigated the effects of estrogen on NER for ultraviolet-induced DNA damage. The N2a neuroblastoma cell line was used as a representative of neuronal cells and 17p-estradiol was selected as one of the most active estrogen derivatives. There were no significant effects of 17p-estradiol on prevention of DNA damage, promotion of DNA repair, or cell survival at the concentration of 0-0.1 microM 17p-estradiol (below cytotoxicity level). These results described that estrogen might not directly affect NER except through another DNA repair system.

  6. Universal Readers Based on Hydrogen Bonding or π-π Stacking for Identification of DNA Nucleotides in Electron Tunnel Junctions.

    PubMed

    Biswas, Sovan; Sen, Suman; Im, JongOne; Biswas, Sudipta; Krstic, Predrag; Ashcroft, Brian; Borges, Chad; Zhao, Yanan; Lindsay, Stuart; Zhang, Peiming

    2016-12-27

    A reader molecule, which recognizes all the naturally occurring nucleobases in an electron tunnel junction, is required for sequencing DNA by a recognition tunneling (RT) technique, referred to as a universal reader. In the present study, we have designed a series of heterocyclic carboxamides based on hydrogen bonding and a large-sized pyrene ring based on a π-π stacking interaction as universal reader candidates. Each of these compounds was synthesized to bear a thiolated linker for attachment to metal electrodes and examined for their interactions with naturally occurring DNA nucleosides and nucleotides by (1)H NMR, ESI-MS, computational calculations, and surface plasmon resonance. RT measurements were carried out in a scanning tunnel microscope. All of these molecules generated electrical signals with DNA nucleotides in tunneling junctions under physiological conditions (phosphate buffered aqueous solution, pH 7.4). Using a support vector machine as a tool for data analysis, we found that these candidates distinguished among naturally occurring DNA nucleotides with the accuracy of pyrene (by π-π stacking interactions) > azole carboxamides (by hydrogen-bonding interactions). In addition, the pyrene reader operated efficiently in a larger tunnel junction. However, the azole carboxamide could read abasic (AP) monophosphate, a product from spontaneous base hydrolysis or an intermediate of base excision repair. Thus, we envision that sequencing DNA using both π-π stacking and hydrogen-bonding-based universal readers in parallel should generate more comprehensive genome sequences than sequencing based on either reader molecule alone.

  7. DNA nanostructure-based imaging probes and drug carriers.

    PubMed

    Zhan, Pengfei; Jiang, Qiao; Wang, Zhen-Gang; Li, Na; Yu, Haiyin; Ding, Baoquan

    2014-09-01

    Self-assembled DNA nanostructures are well-defined nanoscale shapes, with uniform sizes, precise spatial addressability, and excellent biocompatibility. With these features, DNA nanostructures show great potential for biomedical applications; various DNA-based biomedical imaging probes or payload delivery carriers have been developed. In this review, we summarize the recent developments of DNA-based nanostructures as tools for diagnosis and cancer therapy. The biological effects that are brought about by DNA nanostructures are highlighted by in vitro and in vivo imaging, antitumor drug delivery, and immunostimulatory therapy. The challenges and perspectives of DNA nanostructures in the field of nanomedicine are discussed.

  8. Clostridium acetobutylicum 8-Oxoguanine DNA Glycosylase (Ogg) Differs from Eukaryotic Oggs with Respect to Opposite Base Discrimination†

    PubMed Central

    Robey-Bond, Susan M.; Barrantes-Reynolds, Ramiro; Bond, Jeffrey P.; Wallace, Susan S.; Bandaru, Viswanath

    2008-01-01

    During repair of damaged DNA, the oxidized base 8-oxoguanine (8-oxoG) is removed by 8-oxoguanine—DNA glycosylase (Ogg) in eukaryotes and most archaea, whereas in most bacteria it is removed by formamidopyrimidine—DNA glycosylase (Fpg). We report the first characterization of a bacterial Ogg, Clostridium acetobutylicum Ogg (CacOgg). Like human OGG1 and Escherichia coli Fpg (EcoFpg), CacOgg excised 8-oxoguanine. However, unlike hOGG1 and EcoFpg, CacOgg showed little preference for the base opposite the damage during base excision and removed 8-oxoguanine from single-stranded DNA. Thus, our results showed unambiguous qualitative functional differences in vitro between CacOgg and both hOGG1 and EcoFpg. CacOgg differs in sequence from the eukaryotic enzymes at two sequence positions, M132 and F179, which align with amino acids (R154 and Y203) in human OGG1 (hOGG1) found to be involved in opposite base interaction. To address the sequence basis for functional differences with respect to opposite base interactions, we prepared three CacOgg variants, M132R, F179Y, and M132R/F179Y. All three variants showed a substantial increase in specificity for 8-oxoG·C relative to 8-oxoG·A. While we were unable to definitively associate these qualitative functional differences with differences in selective pressure between eukaryotes, Clostridia, and other bacteria, our results are consistent with the idea that evolution of Ogg function is based on kinetic control of repair. PMID:18578506

  9. Induction of DNA strand breaks, base lesions and clustered damage sites in hydrated plasmid DNA films by ultrasoft X rays around the phosphorus K edge.

    PubMed

    Yokoya, Akinari; Cunniffe, Siobhan M T; Watanabe, Ritsuko; Kobayashi, Katsumi; O'Neill, Peter

    2009-09-01

    To characterize the DNA damage induced by K-shell ionization of phosphorus atom in DNA backbone on the level of hydration, the yields of DNA strand breaks and base lesions arising from the interaction of ultrasoft X rays with energies around the phosphorus K edge were determined using dry and fully hydrated pUC18 plasmid DNA samples. Base lesions and bistranded clustered DNA damage sites were revealed by postirradiation treatment with the base excision repair proteins endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of prompt single-strand breaks (SSBs) with dry DNA irradiated at the phosphorus K resonance energy (2153 eV) is about one-third that below the phosphorus K edge (2147 eV). The yields of prompt double-strand breaks (DSBs) were found to be less dependent on the X-ray energy, with the yields being about two times lower when irradiated at 2153 eV. Heat-labile sites were not produced in detectable amounts. The yields of base lesions were dependent on the energy of the X rays, especially when the DNA was fully hydrated. Bistranded clustered DNA damage sites, revealed enzymatically as additional DSBs, were produced in dry as well as in hydrated DNA with all three energies of X rays. The yields of these enzyme-sensitive sites were also lower when irradiated at the phosphorus K resonance energy. On the other hand, the yields of prompt SSBs and enzyme-sensitive sites for the two off-resonance energies were, larger than those determined previously for gamma radiation. The results indicate that the photoelectric effect caused by X rays and dense ionization and excitation events along the tracks of low-energy secondary electrons are more effective at inducing SSBs and enzyme-sensitive sites. The complex types of damage, prompt and enzymatically induced DSBs, are preferentially induced by phosphorus K resonance at 2153 eV rather than simple SSBs and isolated base lesions, particularly in hydrated conditions. It is concluded that not

  10. Active DNA Demethylation in Plants and Animals

    PubMed Central

    Zhang, H.; Zhu, J.-K.

    2013-01-01

    Active DNA demethylation regulates many vital biological processes, including early development and locus-specific gene expression in plants and animals. In Arabidopsis, bifunctional DNA glycosylases directly excise the 5-methylcytosine base and then cleave the DNA backbone at the abasic site. Recent evidence suggests that mammals utilize DNA glycosylases after 5-methylcytosine is oxidized and/or deaminated. In both cases, the resultant single-nucleotide gap is subsequently filled with an unmodified cytosine through the DNA base excision repair pathway. The enzymatic removal of 5-methylcytosine is tightly integrated with histone modifications and possibly noncoding RNAs. Future research will increase our understanding of the mechanisms and critical roles of active DNA demethylation in various cellular processes as well as inspire novel genetic and chemical therapies for epigenetic disorders. PMID:23197304

  11. Breast cancer resistance protein (BCRP) and excision repair cross complement-1 (ERCC1) expression in esophageal cancers and response to cisplatin and irinotecan based chemotherapy

    PubMed Central

    Bharthuar, Anubha; Black, Jennifer D.; Levea, Charles; Malhotra, Usha; Mashtare, Terry L.; Iyer, Renuka

    2014-01-01

    Background Esophageal cancer patients face a dismal outcome despite tri-modality management and median survival remains 15-18 months. Breast cancer resistance protein (BCRP) is an ATP-dependent efflux protein associated with chemotherapy resistance. The role of BCRP expression in esophageal cancer and normal esophageal cells is not known. Excision repair cross complement-1 (ERCC1) overexpression has been correlated with poorer response to cisplatin based chemotherapy. We examined the expression of BCRP and ERCC1 in patients with esophageal cancer and correlated it with survival in patients receiving irinotecan and cisplatin based chemotherapy. Methods With IRB approval, 40 cases of esophageal cancer diagnosed from 2004-2008, were stained for BCRP and ERCC1 expression by immunohistochemistry and scored by a pathologist blinded to clinical data. Baseline demographics, therapy given and survival data were collected and correlated with BCRP and ERCC1 expression. Fisher’s exact test was used to determine association between BCRP and ERCC1 expression and demographics. Cox proportional hazards model was used for association of BCRP and ERCC1 with survival. Results On immunohistochemistry, 30/40 cancers (75%) expressed BCRP. Interestingly, down-regulation of BCRP expression in tumor compared with normal cells was seen in 40% of patients. ERCC1 positivity was seen in 15/30 cases (50%). Median overall survival (OS) was 19 months with no difference in survival between BCRP positive and negative patients (P=0.13) or ERCC1 positive and negative patients (P=0.85). Estimated hazard ratio (HR) of death for BRCP positive patients was 2.29 (95% CI: 0.79-6.64) and for ERCC1 positive patients was 1.09 (95% CI: 0.46-2.56). There was no association of BCRP and ERCC1 expression with disease stage, age, gender or histology. For patients who received cisplatin and irinotecan as first line chemotherapy, there was no difference in survival based on BCRP or ERCC1 status. Conclusions BCRP

  12. FRET based characterization of DNA-based assemblies

    NASA Astrophysics Data System (ADS)

    Buckhout-White, Susan; Gray, Rochester; Ancona, Mario; Goldman, Ellen R.; Medintz, Igor L.

    2014-05-01

    The "spectroscopic ruler" based on fluorescence resonance energy transfer (FRET) is explored as a method for detailed structural characterization of DNA nanostructures in solution. The approach is most directly useful for assessing the positional relationships among chromophores organized by the DNA, but it can also be used to characterize the geometry and kinematics of the DNA scaffold itself. By accumulating data for the distances separating various donor-acceptor pairs, and correlating them with the expected distances, one can quantify the shape and deformability of the structure. A 8x16nm "mini-origami" rectangle is used as the model test structure and the dye-pairs are chosen to investigate anisotropy in the origami's mechanical properties. Not unexpectedly, our analysis finds a strong anisotropy in the stiffness, with the measured spacing across the origami weave deviating much more from expectation than the spacing aligned along the weave pattern.

  13. Mammalian transcription-coupled excision repair.

    PubMed

    Vermeulen, Wim; Fousteri, Maria

    2013-08-01

    Transcriptional arrest caused by DNA damage is detrimental for cells and organisms as it impinges on gene expression and thereby on cell growth and survival. To alleviate transcriptional arrest, cells trigger a transcription-dependent genome surveillance pathway, termed transcription-coupled nucleotide excision repair (TC-NER) that ensures rapid removal of such transcription-impeding DNA lesions and prevents persistent stalling of transcription. Defective TC-NER is causatively linked to Cockayne syndrome, a rare severe genetic disorder with multisystem abnormalities that results in patients' death in early adulthood. Here we review recent data on how damage-arrested transcription is actively coupled to TC-NER in mammals and discuss new emerging models concerning the role of TC-NER-specific factors in this process.

  14. Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epitehlial cells and in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Host DNA damage and DNA repair response to bacterial infections and its significance are not fully understood. Here, we demonstrate that infection by Gram-negative bacterium P. aeruginosa significantly altered the expression and enzymatic activity of base excision DNA repair protein OGG1 in lung epi...

  15. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis.

    PubMed

    Zeng, Zhenshun; Liu, Xiaoxiao; Yao, Jianyun; Guo, Yunxue; Li, Baiyuan; Li, Yangmei; Jiao, Nianzhi; Wang, Xiaoxue

    2016-12-01

    Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation.

  16. Cold adaptation regulated by cryptic prophage excision in Shewanella oneidensis

    PubMed Central

    Zeng, Zhenshun; Liu, Xiaoxiao; Yao, Jianyun; Guo, Yunxue; Li, Baiyuan; Li, Yangmei; Jiao, Nianzhi; Wang, Xiaoxue

    2016-01-01

    Among the environmental stresses experienced by bacteria, temperature shifts are one of the most important. In this study, we discovered a novel cold adaptation mechanism in Shewanella oneidensis that occurs at the DNA level and is regulated by cryptic prophage excision. Previous studies on bacterial cold tolerance mainly focus on the structural change of cell membrane and changes at the RNA and protein levels. Whether or not genomic change can also contribute to this process has not been explored. Here we employed a whole-genome deep-sequencing method to probe the changes at DNA level in a model psychrotrophic bacteria strain. We found that temperature downshift induced a 10 000-fold increase of the excision of a novel P4-like cryptic prophage. Importantly, although prophage excision only occurred in a relatively small population of bacteria, it was able to facilitate biofilm formation and promote the survival of the entire population. This prophage excision affected cell physiology by disrupting a critical gene encoding transfer-messenger RNA (tmRNA). In addition, we found that the histone-like nucleoid-structuring protein (H-NS) could silence prophage excision via binding to the promoter of the putative excisionase gene at warm temperatures. H-NS level was reduced at cold temperatures, leading to de-repression of prophage excision. Collectively, our results reveal that cryptic prophage excision acts as a regulatory switch to enable the survival of the host at low temperature by controlling the activity of tmRNA and biofilm formation. PMID:27482926

  17. Regulation of endonuclease activity in human nucleotide excision repair

    PubMed Central

    Fagbemi, Adebanke F.; Orelli, Barbara; Schärer, Orlando D.

    2011-01-01

    Nucleotide excision repair (NER) is a DNA repair pathway that is responsible for removing a variety of lesions caused by harmful UV light, chemical carcinogens, and environmental mutagens from DNA. NER involves the concerted action of over 30 proteins that sequentially recognize a lesion, excise it in the form of an oligonucleotide, and fill in the resulting gap by repair synthesis. ERCC1-XPF and XPG are structure-specific endonucleases responsible for carrying out the incisions 5′ and 3′ to the damage respectively, culminating in the release of the damaged oligonucleotide. This review focuses on the recent work that led to a greater understanding of how the activities of ERCC1-XPF and XPG are regulated in NER to prevent unwanted cuts in DNA or the persistence of gaps after incision that could result in harmful, cytotoxic DNA structures. PMID:21592868

  18. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

    PubMed Central

    Sozhamannan, Shanmuga; Chute, Michael D; McAfee, Farrell D; Fouts, Derrick E; Akmal, Arya; Galloway, Darrell R; Mateczun, Alfred; Baillie, Leslie W; Read, Timothy D

    2006-01-01

    Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell). Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production. PMID:16600039

  19. On the role of DNA in DNA-based catalytic enantioselective conjugate addition reactions.

    PubMed

    Dijk, Ewold W; Boersma, Arnold J; Feringa, Ben L; Roelfes, Gerard

    2010-09-07

    A kinetic study of DNA-based catalytic enantioselective Friedel-Crafts alkylation and Michael addition reactions showed that DNA affects the rate of these reactions significantly. Whereas in the presence of DNA, a large acceleration was found for the Friedel-Crafts alkylation and a modest acceleration in the Michael addition of dimethyl malonate, a deceleration was observed when using nitromethane as nucleophile. Also, the enantioselectivities proved to be dependent on the DNA sequence. In comparison with the previously reported Diels-Alder reaction, the results presented here suggest that DNA plays a similar role in both cycloaddition and conjugate addition reactions.

  20. Analytical Devices Based on Direct Synthesis of DNA on Paper.

    PubMed

    Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

    2016-01-05

    This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

  1. New design of nucleotide excision repair (NER) inhibitors for combination cancer therapy.

    PubMed

    Gentile, Francesco; Tuszynski, Jack A; Barakat, Khaled H

    2016-04-01

    Many cancer chemotherapy agents act by targeting the DNA of cancer cells, causing substantial damage within their genome and causing them to undergo apoptosis. An effective DNA repair pathway in cancer cells can act in a reverse way by removing these drug-induced DNA lesions, allowing cancer cells to survive, grow and proliferate. In this context, DNA repair inhibitors opened a new avenue in cancer treatment, by blocking the DNA repair mechanisms from removing the chemotherapy-mediated DNA damage. In particular, the nucleotide excision repair (NER) involves more than thirty protein-protein interactions and removes DNA adducts caused by platinum-based chemotherapy. The excision repair cross-complementation group 1 (ERCC1)-xeroderma pigmentosum, complementation group A (XPA) protein (XPA-ERCC1) complex seems to be one of the most promising targets in this pathway. ERCC1 is over expressed in cancer cells and the only known cellular function so far for XPA is to recruit ERCC1 to the damaged point. Here, we build upon our recent advances in identifying inhibitors for this interaction and continue our efforts to rationally design more effective and potent regulators for the NER pathway. We employed in silico drug design techniques to: (1) identify compounds similar to the recently discovered inhibitors, but more effective at inhibiting the XPA-ERCC1 interactions, and (2) identify different scaffolds to develop novel lead compounds. Two known inhibitor structures have been used as starting points for two ligand/structure-hybrid virtual screening approaches. The findings described here form a milestone in discovering novel inhibitors for the NER pathway aiming at improving the efficacy of current platinum-based therapy, by modulating the XPA-ERCC1 interaction.

  2. A Novel DNA-Based Vaccine Methodology for Aids

    DTIC Science & Technology

    1998-11-01

    A., Beck, T.W., Grant, R.F., Bischofberger, N., Benveniste, R.E., Black, R. 1995. Prevention of SIV infection in macaques by (R)-9-(2...enhancement of CTL. Gene gun-based DNA immunization of rhesus macaques resulted in further refinement of DNA delivery parameters based on the degree of...responses by gene gun-based DNA immunization. In addition, we initiated 2 challenge studies in the SIV macaque model to test the role of CTL and mucosal

  3. DNA vs. mirror-image DNA: a universal approach to tune the absolute configuration in DNA-based asymmetric catalysis.

    PubMed

    Wang, Jocelyn; Benedetti, Erica; Bethge, Lucas; Vonhoff, Stefan; Klussmann, Sven; Vasseur, Jean-Jacques; Cossy, Janine; Smietana, Michael; Arseniyadis, Stellios

    2013-10-25

    Mirror mirror on the wall: By taking advantage of the unique structural features of L-DNA, the first examples of left-helical enantioselective induction in the field of DNA-based asymmetric catalysis were realized. Most importantly, this approach is the only one that allows a reliable and predictable access to both enantiomers for any given reaction.

  4. Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy.

    PubMed

    Barhoumi, Aoune; Halas, Naomi J

    2011-12-15

    Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics.

  5. How to make DNA count: DNA-based diagnostic tools in veterinary parasitology.

    PubMed

    Hunt, P W; Lello, J

    2012-05-04

    Traditional methods for the diagnosis of parasitic helminth infections of livestock have a number of limitations, such as the inability to distinguish mixed-species infections, a heavy reliance on technical experience and also sub-sampling errors. Some of these limitations may be overcome through the development of rapid and accurate DNA-based tests. For example, DNA-based tests can specifically detect individual species in a mixed infection at either the larval or egg stages, in the absence of morphological differences among species. Even so, some diagnostic problems remain the same, irrespective of whether a DNA-based or traditional method is used. For example, sub-sampling errors from an aggregated distribution are likely to persist. It is proposed, however, that DNA-based diagnostic technologies offer an opportunity to expand diagnostic capabilities, and are discussed in the current review. The future introduction of DNA-based diagnostic technologies into routine diagnostic settings will also be discussed.

  6. Databases and Bioinformatics Tools for the Study of DNA Repair

    PubMed Central

    Milanowska, Kaja; Rother, Kristian; Bujnicki, Janusz M.

    2011-01-01

    DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions. PMID:22091405

  7. A novel bio-sensor based on DNA strand displacement.

    PubMed

    Shi, Xiaolong; Wang, Zhiyu; Deng, Chenyan; Song, Tao; Pan, Linqiang; Chen, Zhihua

    2014-01-01

    DNA strand displacement technology performs well in sensing and programming DNA segments. In this work, we construct DNA molecular systems based on DNA strand displacement performing computation of logic gates. Specifically, a class of so-called "DNA neurons" are achieved, in which a "smart" way inspired by biological neurons encoding information is developed to encode and deliver information using DNA molecules. The "DNA neuron" is bistable, that is, it can sense DNA molecules as input signals, and release "negative" or "positive" signals DNA molecules. We design intelligent DNA molecular systems that are constructed by cascading some particularly organized "DNA neurons", which could perform logic computation, including AND, OR, XOR logic gates, automatically. Both simulation results using visual DSD (DNA strand displacement) software and experimental results are obtained, which shows that the proposed systems can detect DNA signals with high sensitivity and accretion; moreover, the systems can process input signals automatically with complex nonlinear logic. The method proposed in this work may provide a new way to construct a sensitive molecular signal detection system with neurons spiking behavior in vitro, and can be used to develop intelligent molecular processing systems in vivo.

  8. A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments

    PubMed Central

    Natarajan, Vengadesh Perumal; Zhang, Xinxu; Morono, Yuki; Inagaki, Fumio; Wang, Fengping

    2016-01-01

    Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment. PMID:27446026

  9. Investigation of a Sybr-Green-Based Method to Validate DNA Sequences for DNA Computing

    DTIC Science & Technology

    2005-05-01

    stranded DNA . We previously demonstrated that this technique can be exploited to distinguish between stably-hybridized Watson - Crick duplexes and...et al., 2004) we described the difference between the canonical Watson - Crick base pairs of DNA and the usually less stable mismatches that can also...computing, cross-hybridized duplexes represent errors. It is therefore crucial that DNA sequences be designed so that the formation of a Watson - Crick

  10. Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5'-GTgG-3' sequence: destabilization of two base pairs at the lesion site.

    PubMed

    Brown, Kyle L; Roginskaya, Marina; Zou, Yue; Altamirano, Alvin; Basu, Ashis K; Stone, Michael P

    2010-01-01

    The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson-Crick alignment. The Tg N3H imine and A N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) group is favored; propeller twisting of the Tg*A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G*C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

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

    PubMed

    Chan, Kin; Sterling, Joan F; Roberts, Steven A; Bhagwat, Ashok S; Resnick, Michael A; Gordenin, Dmitry A

    2012-01-01

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

  12. Structure of a DNA glycosylase that unhooks interstrand cross-links.

    PubMed

    Mullins, Elwood A; Warren, Garrett M; Bradley, Noah P; Eichman, Brandt F

    2017-04-10

    DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

  13. An Evolution Based Biosensor Receptor DNA Sequence Generation Algorithm

    PubMed Central

    Kim, Eungyeong; Lee, Malrey; Gatton, Thomas M.; Lee, Jaewan; Zang, Yupeng

    2010-01-01

    A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements. PMID:22315543

  14. Mitochondrial DNA diagnosis for taeniasis and cysticercosis.

    PubMed

    Yamasaki, Hiroshi; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Sato, Marcello Otake; Ito, Akira

    2006-01-01

    Molecular diagnosis for taeniasis and cysticercosis in humans on the basis of mitochondrial DNA analysis was reviewed. Development and application of three different methods, including restriction fragment length polymorphism analysis, base excision sequence scanning thymine-base analysis and multiplex PCR, were described. Moreover, molecular diagnosis of cysticerci found in specimens submitted for histopathology and the molecular detection of taeniasis using copro-DNA were discussed.

  15. Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

    PubMed

    Bennett, Geoffrey R; Peters, Ryan; Wang, Xiao-hong; Hanne, Jeungphill; Sobol, Robert W; Bundschuh, Ralf; Fishel, Richard; Yoder, Kristine E

    2014-01-01

    Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

  16. Controlling charge current through a DNA based molecular transistor

    NASA Astrophysics Data System (ADS)

    Behnia, S.; Fathizadeh, S.; Ziaei, J.

    2017-01-01

    Molecular electronics is complementary to silicon-based electronics and may induce electronic functions which are difficult to obtain with conventional technology. We have considered a DNA based molecular transistor and study its transport properties. The appropriate DNA sequence as a central chain in molecular transistor and the functional interval for applied voltages is obtained. I-V characteristic diagram shows the rectifier behavior as well as the negative differential resistance phenomenon of DNA transistor. We have observed the nearly periodic behavior in the current flowing through DNA. It is reported that there is a critical gate voltage for each applied bias which above it, the electrical current is always positive.

  17. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  18. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  19. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Moyzis, Robert K.; Ratliff, Robert L.; Shera, E. Brooks; Stewart, Carleton C.

    1990-01-01

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

  20. Antibody-controlled actuation of DNA-based molecular circuits

    NASA Astrophysics Data System (ADS)

    Engelen, Wouter; Meijer, Lenny H. H.; Somers, Bram; de Greef, Tom F. A.; Merkx, Maarten

    2017-02-01

    DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody-epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

  1. Application of a Pattern-based Classification System for Invasive Endocervical Adenocarcinoma in Cervical Biopsy, Cone and Loop Electrosurgical Excision (LEEP) Material: Pattern on Cone and LEEP is Predictive of Pattern in the Overall Tumor.

    PubMed

    Djordjevic, Bojana; Parra-Herran, Carlos

    2016-09-01

    A pattern-based classification system has been recently proposed for invasive endocervical adenocarcinoma, which is predictive of the risk of nodal metastases. Identifying cases at risk of nodal involvement is most relevant at the time of biopsy and loop electrosurgical excision procedure (LEEP) to allow for optimal surgical planning, and, most importantly, consideration of lymphadenectomy. This study aims to determine the topography of patterns of stromal invasion in invasive endocervical adenocarcinoma with emphasis on patterns in biopsy, cone, and LEEP. Invasive pattern was assessed following the pattern-based classification (Patterns A, B, and C) in 47 invasive endocervical adenocarcinomas treated with hysterectomy or trachelectomy and correlated with pattern of invasion at the tumor surface (2 mm of tumor depth) and on preoperative biopsy and cone/LEEP. Patterns A, B, and C were present in 21.3%, 36.2%, and 42.5% of cases, respectively. Most pattern A cases were Stage IA (90%), whereas most Pattern B and C cases were Stage IB (76.5% and 80%, respectively). Horizontal spread was on average larger in Pattern C (24.1 mm) than in Patterns A and B (7.7 and 12.3 mm, respectively). Pattern at the tumor surface correlated with the overall pattern in 95.7% of cases. Concordance between patterns at cone/LEEP and hysterectomy was 92.8%; the only discrepant case was upgraded from Pattern A on LEEP to C on final excision. Agreement between patterns in biopsy and the overall tumor, however, was only 37.5%. In all discrepant cases, biopsy failed to reveal destructive invasion, which was evident on excision. All discrepant biopsies with pattern A showed glandular complexity resembling exophytic papillary growth but did not meet criteria for destructive invasion. On excision, marked gland confluence with papillary architecture was evident. We conclude that the pattern of invasion on cone/LEEP is a good predictor of pattern of invasion on hysterectomy, particularly if there is

  2. Reflective type objective based spectral-domain phase-sensitive optical coherence tomography for high-sensitive structural and functional imaging of cochlear microstructures through intact bone of an excised guinea pig cochlea

    NASA Astrophysics Data System (ADS)

    Subhash, Hrebesh M.; Wang, Ruikang K.; Chen, Fangyi; Nuttall, Alfred L.

    2013-03-01

    Most of the optical coherence tomographic (OCT) systems for high resolution imaging of biological specimens are based on refractive type microscope objectives, which are optimized for specific wave length of the optical source. In this study, we present the feasibility of using commercially available reflective type objective for high sensitive and high resolution structural and functional imaging of cochlear microstructures of an excised guinea pig through intact temporal bone. Unlike conventional refractive type microscopic objective, reflective objective are free from chromatic aberrations due to their all-reflecting nature and can support a broadband of spectrum with very high light collection efficiency.

  3. Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

    PubMed

    Guo, Xiaoge; Jinks-Robertson, Sue

    2013-12-01

    Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral

  4. Forensic aspects of DNA-based human identity testing.

    PubMed

    Roper, Stephen M; Tatum, Owatha L

    2008-01-01

    The forensic applications of DNA-based human identity laboratory testing are often underappreciated. Molecular biology has seen an exponential improvement in the accuracy and statistical power provided by identity testing in the past decade. This technology, dependent upon an individual's unique DNA sequence, has cemented the use of DNA technology in the forensic laboratory. This paper will discuss the state of modern DNA-based identity testing, describe the technology used to perform this testing, and describe its use as it relates to forensic applications. We will also compare individual technologies, including polymerase chain reaction (PCR) and Southern Blotting, that are used to detect the molecular differences that make all individuals unique. An increasing reliance on DNA-based identity testing dictates that healthcare providers develop an understanding of the background, techniques, and guiding principles of this important forensic tool.

  5. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    SciTech Connect

    Jackson, Christopher B.; Gallati, Sabina; Schaller, Andre

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  6. DNA tile based self-assembly: building complex nanoarchitectures.

    PubMed

    Lin, Chenxiang; Liu, Yan; Rinker, Sherri; Yan, Hao

    2006-08-11

    DNA tile based self-assembly provides an attractive route to create nanoarchitectures of programmable patterns. It also offers excellent scaffolds for directed self-assembly of nanometer-scale materials, ranging from nanoparticles to proteins, with potential applications in constructing nanoelectronic/nanophotonic devices and protein/ligand nanoarrays. This Review first summarizes the currently available DNA tile toolboxes and further emphasizes recent developments toward self-assembling DNA nanostructures with increasing complexity. Exciting progress using DNA tiles for directed self-assembly of other nanometer scale components is also discussed.

  7. DNA-based cryptographic methods for data hiding in DNA media.

    PubMed

    Marwan, Samiha; Shawish, Ahmed; Nagaty, Khaled

    2016-12-01

    Information security can be achieved using cryptography, steganography or a combination of them, where data is firstly encrypted using any of the available cryptography techniques and then hid into any hiding medium. Recently, the famous genomic DNA has been introduced as a hiding medium, known as DNA steganography, due to its notable ability to hide huge data sets with a high level of randomness and hence security. Despite the numerous cryptography techniques, to our knowledge only the vigenere cipher and the DNA-based playfair cipher have been combined with the DNA steganography, which keeps space for investigation of other techniques and coming up with new improvements. This paper presents a comprehensive analysis between the DNA-based playfair, vigenere, RSA and the AES ciphers, each combined with a DNA hiding technique. The conducted analysis reports the performance diversity of each combined technique in terms of security, speed, hiding capacity in addition to both key size and data size. Moreover, this paper proposes a modification of the current combined DNA-based playfair cipher technique, which makes it not only simple and fast but also provides a significantly higher hiding capacity and security. The conducted extensive experimental studies confirm such outstanding performance in comparison with all the discussed combined techniques.

  8. DNA-based computing of strategic assignment problems.

    PubMed

    Shu, Jian-Jun; Wang, Qi-Wen; Yong, Kian-Yan

    2011-05-06

    DNA-based computing is a novel technique to tackle computationally difficult problems, in which computing time grows exponentially corresponding to problematic size. A strategic assignment problem is a typical nondeterministic polynomial problem, which is often associated with strategy applications. In this Letter, a new approach dealing with strategic assignment problems is proposed based on manipulating DNA strands, which is believed to be better than the conventional silicon-based computing in solving the same problem.

  9. Therapeutic option of plasmid-DNA based gene transfer.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Kunugiza, Yasuo; Iekushi, Kazuma; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

  10. Unique magnetic signatures of mismatched base pairs in DNA

    NASA Astrophysics Data System (ADS)

    Apalkov, Vadim; Berashevich, Julia; Chakraborty, Tapash

    2010-02-01

    Magnetic properties of DNA containing mispairs, such as different conformations of the GṡA mispair, or a GṡT mispair inserted into the DNA chain, have been theoretically investigated. The essential ingredients for these studies, the charge transfer integrals, were evaluated from the DNA sequences containing the mispair and optimized in the solvent. We find that the magnetic susceptibilities of the host DNA chain containing a large number of Watson-Crick base pairs are significantly altered in the presence of the mispairs, and the effects depend on the choice of mispairs. In particular, insertion of even a single GṡA mispair changes the nature of magnetization (sign of the susceptibility) of the host DNA. We propose that measurement of the magnetic properties of DNA might provide a direct route to detection and identification of those mispairs.

  11. Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination.

    PubMed

    Henley, Robert Y; Vazquez-Pagan, Ana G; Johnson, Michael; Kanavarioti, Anastassia; Wanunu, Meni

    2015-01-01

    Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation.

  12. Integrating DNA-based data into bioassessments improves ...

    EPA Pesticide Factsheets

    The integration of DNA-based identification methods into bioassessments could result in more accurate representations of species distributions and species-habitat relationships. DNA-based approaches may be particularly informative for tracking the distributions of rare and/or invasive species that can comprise a small proportion of samples or are difficult to identify morphologically. In 2012 and 2013, we used a combination of morphological and DNA-based methods (meta-barcoding) to identify fish eggs and larvae collected in the St. Louis River estuary area, Minnesota. We found a large proportion of cases where a lack of agreement occurred between species identified at a site using morphological versus DNA identification, prompting a discussion of how to best reconcile these differences. Choices made during sampling collection, DNA amplification/extraction, and bioinformatics processing influence the DNA-morphology match. The distribution of some species (including several invasives) and their relationships to habitat changed after DNA-data was incorporated. Results highlight how incorporating of DNA-data may get us closer to the “truth”, which has large ramifications in the search for rare species and when understanding the environmental drivers of species distributions is important for management. not applicable

  13. OGG1-DNA interactions facilitate NF-κB binding to DNA targets

    PubMed Central

    Pan, Lang; Hao, Wenjing; Zheng, Xu; Zeng, Xianlu; Ahmed Abbasi, Adeel; Boldogh, Istvan; Ba, Xueqing

    2017-01-01

    DNA repair protein counteracting oxidative promoter lesions may modulate gene expression. Oxidative DNA bases modified by reactive oxygen species (ROS), primarily as 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair (BER) pathway. Because cellular response to oxidative challenge is accompanied by DNA damage repair, we tested whether the repair by OGG1 is compatible with transcription factor binding and gene expression. We performed electrophoretic mobility shift assay (EMSA) using wild-type sequence deriving from Cxcl2 gene promoter and the same sequence bearing a single synthetic 8-oxoG at defined 5′ or 3′ guanine in runs of guanines to mimic oxidative effects. We showed that DNA occupancy of NF-κB present in nuclear extracts from tumour necrosis factor alpha (TNFα) exposed cells is OGG1 and 8-oxoG position dependent, importantly, OGG1 counteracting 8-oxoG outside consensus motif had a profound influence on purified NF-κB binding to DNA. Furthermore, OGG1 is essential for NF-κB dependent gene expression, prior to 8-oxoG excised from DNA. These observations imply that pre-excision step(s) during OGG1 initiated BER evoked by ROS facilitates NF-κB DNA occupancy and gene expression. PMID:28266569

  14. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    PubMed

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

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

    SciTech Connect

    Moscariello, M.M.; Sutherland, B.

    2010-08-01

    DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents.

  16. Stacking with the unnatural DNA base 6-ethynylpyridone

    NASA Astrophysics Data System (ADS)

    Gibson, Douglas J.; van Mourik, Tanja

    2017-01-01

    It was previously reported that the incorporation of 6-ethynylpyridone (E) into a DNA duplex (replacing T in a T:A base pair) leads to DNA duplexes that are more stable than the T:A-containing duplexes. DFT calculations at the M06-2X/6-31+G(d) and BLYP-D3/6-31+G(d) levels on various base pairs, stacked bases and stacked base pairs in continuum solvation water suggest that the observed increased stability of E:A-containing duplexes is due to the combined effects of stronger base pairing and enhanced stacking of the E:A base pair.

  17. A liquid-crystal-based DNA biosensor for pathogen detection

    NASA Astrophysics Data System (ADS)

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-03-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection.

  18. DNA-based self-assembly for functional nanomaterials.

    PubMed

    Wang, Zhen-Gang; Ding, Baoquan

    2013-07-26

    The unprecedented development of DNA nanotechnology has caused DNA self-assembly to attract close attention in many disciplines. In this research news article, the employment of DNA self-assembly in the fields of materials science and nanotechnology is described. DNA self-assembly can be used to prepare bulk-scale hydrogels and 3D macroscopic crystals with nanoscale internal structures, to induce the crystallization of nanoparticles, to template the fabrication of organic conductive nanomaterials, and to act as drug delivery vehicles for therapeutic agents. The properties and functions are fully tunable because of the designability and specificity of DNA assembly. Moreover, because of the intrinsic dynamics, DNA self-assembly can act as a program switch and can efficiently control stimuli responsiveness. We highlight the power of DNA self-assembly in the preparation and function regulation of materials, aiming to motivate future multidisciplinary and interdisciplinary research. Finally, we describe some of the challenges currently faced by DNA assembly that may affect the functional evolution of such materials, and we provide our insights into the future directions of several DNA self-assembly-based nanomaterials.

  19. A liquid-crystal-based DNA biosensor for pathogen detection

    PubMed Central

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-01-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection. PMID:26940532

  20. Multiple Base Substitution Corrections in DNA Sequence Evolution

    NASA Astrophysics Data System (ADS)

    Kowalczuk, M.; Mackiewicz, P.; Szczepanik, D.; Nowicka, A.; Dudkiewicz, M.; Dudek, M. R.; Cebrat, S.

    We discuss the Jukes and Cantor's one-parameter model and Kimura's two-parameter model unability to describe evolution of asymmetric DNA molecules. The standard distance measure between two DNA sequences, which is the number of substitutions per site, should include the effect of multiple base substitutions separately for each type of the base. Otherwise, the respective tables of substitutions cannot reconstruct the asymmetric DNA molecule with respect to the composition. Basing on Kimura's neutral theory, we have derived a linear law for the correlation of the mean survival time of nucleotides under constant mutation pressure and their fraction in the genome. According to the law, the corrections to Kimura's theory have been discussed to describe evolution of genomes with asymmetric nucleotide composition. We consider the particular case of the strongly asymmetric Borrelia burgdorferi genome and we discuss in detail the corrections, which should be introduced into the distance measure between two DNA sequences to include multiple base substitutions.

  1. HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β

    PubMed Central

    Fang, Qingming; Inanc, Burcu; Schamus, Sandy; Wang, Xiao-hong; Wei, Leizhen; Brown, Ashley R.; Svilar, David; Sugrue, Kelsey F.; Goellner, Eva M.; Zeng, Xuemei; Yates, Nathan A.; Lan, Li; Vens, Conchita; Sobol, Robert W.

    2014-01-01

    Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance. PMID:25423885

  2. Operation of a DNA-Based Autocatalytic Network in Serum

    NASA Astrophysics Data System (ADS)

    Graugnard, Elton; Cox, Amber; Lee, Jeunghoon; Jorcyk, Cheryl; Yurke, Bernard; Hughes, William L.

    The potential for inferring the presence of cancer by the detection of miRNA in human blood has motivated research into the design and operation of DNA-based chemical amplifiers that can operate in bodily fluids. As a first step toward this goal, we have tested the operation of a DNA-based autocatalytic network in human serum and mouse serum. With the addition of sodium dodecyl sulfate to prevent degradation by nuclease activity, the network was found to operate successfully with both DNA and RNA catalysts.

  3. DNA sequence analysis with droplet-based microfluidics

    PubMed Central

    Abate, Adam R.; Hung, Tony; Sperling, Ralph A.; Mary, Pascaline; Rotem, Assaf; Agresti, Jeremy J.; Weiner, Michael A.; Weitz, David A.

    2014-01-01

    Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence. PMID:24185402

  4. Discovery of Benzothiazole Scaffold-Based DNA Gyrase B Inhibitors.

    PubMed

    Gjorgjieva, Marina; Tomašič, Tihomir; Barančokova, Michaela; Katsamakas, Sotirios; Ilaš, Janez; Tammela, Päivi; Peterlin Mašič, Lucija; Kikelj, Danijel

    2016-10-13

    Bacterial DNA gyrase and topoisomerase IV control the topological state of DNA during replication and are validated targets for antibacterial drug discovery. Starting from our recently reported 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-based DNA gyrase B inhibitors, we replaced their central core with benzothiazole-2,6-diamine scaffold and interchanged substituents in positions 2 and 6. This resulted in equipotent nanomolar inhibitors of DNA gyrase from Escherichia coli displaying improved inhibition of Staphylococcus aureus DNA gyrase and topoisomerase IV from both bacteria. Compound 27 was the most balanced inhibitor of DNA gyrase and topoisomerase IV from both E. coli and S. aureus. The crystal structure of the 2-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzothiazol-6-yl)amino)-2-oxoacetic acid (24) in complex with E. coli DNA gyrase B revealed the binding mode of the inhibitor in the ATP-binding pocket. Only some compounds possessed weak antibacterial activity against Gram-positive bacteria. These results provide a basis for structure-based optimization toward dual DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.

  5. Artifacts associated with the measurement of oxidized DNA bases.

    PubMed Central

    Cadet, J; Douki, T; Ravanat, J L

    1997-01-01

    In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay. Images Figure 1. PMID:9349826

  6. Artifacts associated with the measurement of oxidized DNA bases.

    PubMed

    Cadet, J; Douki, T; Ravanat, J L

    1997-10-01

    In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay.

  7. Lower nucleotide excision repair capacity in newborns compared to their mothers: a pilot study.

    PubMed

    Vande Loock, Kim; Decordier, Ilse; Plas, Gina; Ciardelli, Roberta; Haumont, Dominique; Kirsch-Volders, Micheline

    2014-01-01

    Recognition of the potential vulnerability of children and newborns and protection of their health is essential, especially regarding to genotoxic compounds. Benzo(a)pyrene B(a)P a commonly found carcinogen, and its metabolite BPDE, are known to cross the placenta. To investigate how well newborns are able to cope with BPDE-induced DNA damage, a recent developed nucleotide excision repair cell phenotype assay was applied in a pilot study of 25 newborn daughters and their mothers, using the Alkaline Comet Assay and taking demographic data into account. Newborns seemed to be less able to repair BPDE-induced DNA damage since lower repair capacity levels were calculated compared to their mothers although statistical significance was not reached. Assessment of repair capacity in combination with genotypes will provide important information to support preventive strategies in neonatal care and to define science based exposure limits for pregnant women and children.

  8. HO* radicals induce an unexpected high proportion of tandem base lesions refractory to repair by DNA glycosylases.

    PubMed

    Bergeron, François; Auvré, Frédéric; Radicella, J Pablo; Ravanat, Jean-Luc

    2010-03-23

    Reaction of HO(*) radicals with double-stranded calf thymus DNA produces high levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and, to a minor extent, 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodAdo). Formation of the hydroxylated purine lesions is explained by addition of HO(*) to the C8 position of the purine moiety. It has been reported that tandem lesions containing a formylamine residue neighboring 8-oxodGuo could be produced through addition of a transiently generated pyrimidine peroxyl radical onto the C8 of an adjacent purine base. Formation of such tandem lesions accounted for approximately 10% of the total 8-oxodGuo. In the present work we show that addition of HO(*) onto the C8 of purine accounts for only approximately 5% of the generated 8-oxodGuo. About 50% of the 8-hydroxylated purine lesions, including 8-oxodGuo and 8-oxodAdo, are involved in tandem damage and are produced by peroxyl addition onto the C8 of a vicinal purine base. In addition, the remaining 45% of the 8-oxodGuo are produced by an electron transfer reaction, providing an explanation for the higher yield of formation of 8-oxodGuo compared to 8-oxodAdo. Interestingly, we show that >40% of the 8-oxodGuo involved in tandem lesions is refractory to excision by DNA glycosylases. Altogether our results demonstrate that, subsequently to a single oxidation event, peroxidation reactions significantly increase the yield of formation of hydroxylated purine modifications, generating a high proportion of tandem lesions partly refractory to base excision repair.

  9. DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles.

    PubMed

    Liu, Pei; Wang, Dandan; Zhou, Yunlei; Wang, Haiyan; Yin, Huanshun; Ai, Shiyun

    2016-06-15

    DNA methyltransferase (MTase) has received a large amount of attention due to its catalyzation of DNA methylation in both eukaryotes and prokaryotes, which has a close relationship to cancer and bacterial diseases. Herein, a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine (polyA) DNA with a gold nanoparticles functioned glassy carbon electrode (AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue (MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27 U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in real biological matrices and inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery.

  10. A CLIQUE algorithm using DNA computing techniques based on closed-circle DNA sequences.

    PubMed

    Zhang, Hongyan; Liu, Xiyu

    2011-07-01

    DNA computing has been applied in broad fields such as graph theory, finite state problems, and combinatorial problem. DNA computing approaches are more suitable used to solve many combinatorial problems because of the vast parallelism and high-density storage. The CLIQUE algorithm is one of the gird-based clustering techniques for spatial data. It is the combinatorial problem of the density cells. Therefore we utilize DNA computing using the closed-circle DNA sequences to execute the CLIQUE algorithm for the two-dimensional data. In our study, the process of clustering becomes a parallel bio-chemical reaction and the DNA sequences representing the marked cells can be combined to form a closed-circle DNA sequences. This strategy is a new application of DNA computing. Although the strategy is only for the two-dimensional data, it provides a new idea to consider the grids to be vertexes in a graph and transform the search problem into a combinatorial problem.

  11. NMR analysis of base-pair opening kinetics in DNA.

    PubMed

    Szulik, Marta W; Voehler, Markus; Stone, Michael P

    2014-12-12

    Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base-pair opening and closing kinetics of individual double-stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state-of-the art techniques and NMR instrumentation, including cryoprobes, is discussed.

  12. Charge Transport across DNA-Based Three-Way Junctions.

    PubMed

    Young, Ryan M; Singh, Arunoday P N; Thazhathveetil, Arun K; Cho, Vincent Y; Zhang, Yuqi; Renaud, Nicolas; Grozema, Ferdinand C; Beratan, David N; Ratner, Mark A; Schatz, George C; Berlin, Yuri A; Lewis, Frederick D; Wasielewski, Michael R

    2015-04-22

    DNA-based molecular electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across DNA three-way junction (3WJ) have yet to be determined. Here, we present an investigation of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and molecular dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently populated hole carrier nucleobases into better aligned geometries on the nanosecond time scale, thus modulating the π-π electronic coupling along the base pair sequence.

  13. Programmable molecular recognition based on the geometry of DNA nanostructures

    NASA Astrophysics Data System (ADS)

    Woo, Sungwook; Rothemund, Paul W. K.

    2011-08-01

    From ligand-receptor binding to DNA hybridization, molecular recognition plays a central role in biology. Over the past several decades, chemists have successfully reproduced the exquisite specificity of biomolecular interactions. However, engineering multiple specific interactions in synthetic systems remains difficult. DNA retains its position as the best medium with which to create orthogonal, isoenergetic interactions, based on the complementarity of Watson-Crick binding. Here we show that DNA can be used to create diverse bonds using an entirely different principle: the geometric arrangement of blunt-end stacking interactions. We show that both binary codes and shape complementarity can serve as a basis for such stacking bonds, and explore their specificity, thermodynamics and binding rules. Orthogonal stacking bonds were used to connect five distinct DNA origami. This work, which demonstrates how a single attractive interaction can be developed to create diverse bonds, may guide strategies for molecular recognition in systems beyond DNA nanostructures.

  14. The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

    PubMed Central

    Roos, Wynand Paul; Krumm, Andrea

    2016-01-01

    Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization. PMID:27738139

  15. Progression of DNA damage induced by a glyphosate-based herbicide in fish (Anguilla anguilla) upon exposure and post-exposure periods--insights into the mechanisms of genotoxicity and DNA repair.

    PubMed

    Marques, Ana; Guilherme, Sofia; Gaivão, Isabel; Santos, Maria Ana; Pacheco, Mário

    2014-11-01

    Roundup® is a glyphosate-based herbicide widely used with both agricultural and non-agricultural purposes, which has been demonstrated to represent a risk to non-target aquatic organisms, namely fish. Among the described effects to fish, genotoxicity has been pointed out as one of the most hazardous. However, the genotoxic mechanisms of Roundup® as well as the involvement of the oxidative DNA damage repair system are not entirely understood. Hence, this work aimed to improve the knowledge on the progression of DNA damage upon short-term exposure (3 days) and post-exposure (1-14 days) periods in association with DNA repair processes in Anguilla anguilla exposed to Roundup® (58 and 116 μg L⁻¹). DNA damage in hepatic cells was evaluated by the comet assay improved with the DNA-lesion specific endonucleases FPG and EndoIII. In order to evaluate the oxidative DNA damage repair ability, an in vitro base excision repair (BER) assay was performed, testing hepatic subcellular extracts. Besides the confirmation of the genotoxic potential of this herbicide, oxidative damage was implicit as an important mechanism of genetic damage, which showed to be transient, since DNA integrity returned to the control levels on the first day after cessation of exposure. An increased capacity to repair oxidative DNA damage emerging in the post-exposure period revealed to be a crucial pathway for the A. anguilla recovery; nevertheless, DNA repair machinery showed to be susceptible to inhibitory actions during the exposure period, disclosing another facet of the risk associated with the tested agrochemical.

  16. Stability and proton transfer in DNA base pairs of AMD473-DNA adduct

    NASA Astrophysics Data System (ADS)

    Sarmah, Pubalee; Deka, Ramesh C.

    2011-05-01

    We investigate the energetics of four different adducts of cisplatin analogue cis-[PtCl 2(NH 3)(2-picoline)] (AMD473) with a duplex DNA using DFT/ONIOM methods to probe their stabilities. Further, we study the possibilities of proton transfer between DNA base pairs of the most stable drug-DNA adduct. The adduct b(2-picoline trans to 3'-G and 2-methyl group directs to the DNA major groove) is found to be the most stable configuration among all the possible adducts. From the proton transfer analysis we found that the single proton transfer between N1 position of guanine (G) and N3 position of cytosine (C) of each GC pair gives a structure energetically as stable as the original one.

  17. Endoscopic excision of cheek lipomas.

    PubMed

    Pyon, Jai-Kyong; Park, Bum-Jin; Mun, Goo-Hyun; Cha, Myung-Kyu; Lim, So-Young; Bang, Sa-Ik; Oh, Kap-Sung

    2008-10-01

    Although the removal of forehead and brow benign tumors using an endoscopic technique has proven to be valuable, the efficacy of an endoscopic excision for cheek masses is unclear. A retrospective review was performed on 8 patients with a lipoma (7) and a foreign body granuloma (1) located at the cheek region. There were 7 men and 1 woman with a mean age of 34.8 years (range, 22-54 years). All the excisional procedures were performed with an endoscope through 2 small incisions, one on the hair-bearing sideburns and the other behind the earlobe. The masses varied from 0.7 x 0.7 cm to 4.0 x 3.0 cm in size. There were no intraoperative or postoperative complications, and no recurrence was detected after a 5- to 61-month follow-up. An endoscopically assisted excision of cheek lipomas is an effective procedure and might be a good alternative to the more conventional procedures.

  18. Cooperativity-based modeling of heterotypic DNA nanostructure assembly.

    PubMed

    Shapiro, Anastasia; Hozeh, Avital; Girshevitz, Olga; Abu-Horowitz, Almogit; Bachelet, Ido

    2015-07-27

    DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction-in which arms are unequal-as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations.

  19. Cooperativity-based modeling of heterotypic DNA nanostructure assembly

    PubMed Central

    Shapiro, Anastasia; Hozeh, Avital; Girshevitz, Olga; Abu-Horowitz, Almogit; Bachelet, Ido

    2015-01-01

    DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction—in which arms are unequal—as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations. PMID:26071955

  20. DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide Cleavage-Based Electrochemical Biosensor.

    PubMed

    Kou, Bei-Bei; Zhang, Li; Xie, Hua; Wang, Ding; Yuan, Ya-Li; Chai, Ya-Qin; Yuan, Ruo

    2016-09-07

    Herein, we developed a label-free electrochemical biosensor for sensitive detection of matrix metalloproteinase-7 (MMP-7) based on DNA enzyme-decorated DNA nanoladders as enhancer. A peptide and single-stranded DNA S1-modified platinum nanoparticles (P1-PtNPs-S1), which served as recognition nanoprobes, were first immobilized on electrode. When target MMP-7 specifically recognized and cleaved the peptide, the PtNPs-S1 bioconjugates were successfully released from electrode. The remaining S1 on electrode then hybridized with ssDNA1 (I1) and ssDNA2 (I2), which could synchronously trigger two hybridization chain reactions (HCRs), resulting in the in situ formation of DNA nanoladders. The desired DNA nanoladders not only were employed as ideal nanocarriers for enzyme loading, but also maintained its catalytic activity. With the help of hydrogen peroxide (H2O2), manganese porphyrin (MnPP) with peroxidase-like activity accelerated the 4-chloro-1-naphthol (4-CN) oxidation with generation of insoluble precipitation on electrode, causing a very low differential pulse voltammetry (DPV) signal for quantitative determination of MMP-7. Under optimal conditions, the developed biosensor exhibited a wide linear ranging from 0.2 pg/mL to 20 ng/mL, and the detection limit was 0.05 pg/mL. This work successfully realized the combination of DNA signal amplification technique with artificial mimetic enzyme-catalyzed precipitation reaction in peptide cleavage-based protein detection, offering a promising avenue for the detection of other proteases.

  1. Excision repair and patch size in UV-irradiated bacteriophage T4

    SciTech Connect

    Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

    1981-11-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control, experiments with the denV/sub 1/ excision-deificient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  2. Excision repair and patch size in UV-irradiated bacteriophage T4

    SciTech Connect

    Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

    1981-11-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  3. Pure cutting current for loop excision of squamous intraepithelial lesions.

    PubMed

    McLucas, B; McGill, J

    1994-05-01

    Clear margins are critical to the identification of complete excision of premalignant lesions on the cervix. Large loop excision of the transformation zone aids the pathologic evaluation of the excised specimen while it causes minimal thermal damage. Prior studies of loop excision were performed with a cutting current blended with a coagulating waveform to aid hemostasis. Blended current has higher voltage, which may cause tissue to stick to the electrode and produce thermal damage to the cervix. In this series, pure cutting current was used to excise the cervical transformation zone in 20 patients. The depth of thermal damage was studied in 6 patients; the average endocervical zone of damage was 0.47 mm and that of the exocervical zone, 0.43 mm. The base of the cervix could be examined with the colposcope for the presence of glands. None of our procedures was complicated by intraoperative or delayed bleeding. The preoperative injection of a vasoconstrictor into the cervical stroma is thought to aid the surgery by its hemostatic properties.

  4. Responsive DNA-based hydrogels and their applications

    PubMed Central

    Xiong, Xiangling; Zhou, Cuisong; Wu, Cuichen; Zhu, Guizhi; Chen, Zhuo; Tan, Weihong

    2015-01-01

    The term hydrogel describes a type of soft and wet material formed by crosslinked hydrophilic polymers. The distinct feature of hydrogels is their ability to absorb a large amount of water and swell. The properties of a hydrogel are usually determined by the type of polymer and crosslinker, the degree of crosslinking, and the water content. However, a group of hydrogels, called “smart hydrogels”, changes properties in response to environmental changes or external stimuli. Recently, DNA or DNA-inspired responsive hydrogels have attracted considerable attention in construction of smart hydrogels because of the intrinsic advantages of DNA. As a biological polymer, DNA is hydrophilic, biocompatible, and highly programmable by Watson-Crick base pairing. DNA can form a hydrogel by itself under certain conditions, and it can also be incorporated into synthetic polymers to form DNA-hybrid hydrogels. Functional DNAs, such as aptamers and DNAzymes, provide additional molecular recognition capabilities and versatility. In this review, we discuss DNA-based hydrogels in terms of their stimulus response, as well as their applications. PMID:23857726

  5. Excision of the piggyBac transposable element in maize cells is a precise event

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The piggyBac transposable element (TE) from the moth Trichoplusia ni encodes a ‘cut and paste’ DNA transposase that has been used to transform a number of insects, as well as planaria, mammalian cells, and mice. The wild type and a mutated piggyBac TE excised from a DNA vector in transient assays u...

  6. A Novel Image Encryption Algorithm Based on DNA Subsequence Operation

    PubMed Central

    Zhang, Qiang; Xue, Xianglian; Wei, Xiaopeng

    2012-01-01

    We present a novel image encryption algorithm based on DNA subsequence operation. Different from the traditional DNA encryption methods, our algorithm does not use complex biological operation but just uses the idea of DNA subsequence operations (such as elongation operation, truncation operation, deletion operation, etc.) combining with the logistic chaotic map to scramble the location and the value of pixel points from the image. The experimental results and security analysis show that the proposed algorithm is easy to be implemented, can get good encryption effect, has a wide secret key's space, strong sensitivity to secret key, and has the abilities of resisting exhaustive attack and statistic attack. PMID:23093912

  7. An Optimal Seed Based Compression Algorithm for DNA Sequences

    PubMed Central

    Gopalakrishnan, Gopakumar; Karunakaran, Muralikrishnan

    2016-01-01

    This paper proposes a seed based lossless compression algorithm to compress a DNA sequence which uses a substitution method that is similar to the LempelZiv compression scheme. The proposed method exploits the repetition structures that are inherent in DNA sequences by creating an offline dictionary which contains all such repeats along with the details of mismatches. By ensuring that only promising mismatches are allowed, the method achieves a compression ratio that is at par or better than the existing lossless DNA sequence compression algorithms. PMID:27555868

  8. Age dependency of base modification in rabbit liver DNA

    NASA Technical Reports Server (NTRS)

    Yamamoto, O.; Fuji, I.; Yoshida, T.; Cox, A. B.; Lett, J. T.

    1988-01-01

    Age-related modifications of DNA bases have been observed in the liver of the New Zealand white (NZW) rabbit (Oryctolagus cuniculus), a lagomorph with a median life span in captivity of 5-7 yr. The ages of the animals studied ranged from 6 wk to 9 yr. After the DNA had been extracted from the liver cell nuclei and hydrolyzed with acid, the bases were analyzed by column chromatography with Cellulofine gels (GC-15-m). Two peaks in the chromatogram, which eluted before the four DNA bases, contained modified bases. Those materials, which were obtained in relatively large amounts from old animals, were highly fluorescent, and were shown to be crosslinked base products by mass spectrometry. The yield of crosslinked products versus rabbit age (greater than 0.5 yr) can be fitted by an exponential function (correlation coefficient: 0.76 +/- 0.09).

  9. A Rewritable, Random-Access DNA-Based Storage System.

    PubMed

    Yazdi, S M Hossein Tabatabaei; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-09-18

    We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications.

  10. Molecular Dynamics Simulations of Perylenediimide DNA Base Surrogates.

    PubMed

    Markegard, Cade B; Mazaheripour, Amir; Jocson, Jonah-Micah; Burke, Anthony M; Dickson, Mary N; Gorodetsky, Alon A; Nguyen, Hung D

    2015-09-03

    Perylene-3,4,9,10-tetracarboxylic diimides (PTCDIs) are a well-known class of organic materials. Recently, these molecules have been incorporated within DNA as base surrogates, finding ready applications as probes of DNA structure and function. However, the assembly dynamics and kinetics of PTCDI DNA base surrogates have received little attention to date. Herein, we employ constant temperature molecular dynamics simulations to gain an improved understanding of the assembly of PTCDI dimers and trimers. We also use replica-exchange molecular dynamics simulations to elucidate the energetic landscape dictating the formation of stacked PTCDI structures. Our studies provide insight into the equilibrium configurations of multimeric PTCDIs and hold implications for the construction of DNA-inspired systems from perylene-derived organic semiconductor building blocks.

  11. ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion.

    PubMed

    Ponferrada-Marín, María Isabel; Roldán-Arjona, Teresa; Ariza, Rafael R

    2009-07-01

    Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation.

  12. ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion

    PubMed Central

    Ponferrada-Marín, María Isabel; Roldán-Arjona, Teresa; Ariza, Rafael R.

    2009-01-01

    Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation. PMID:19443451

  13. Heterogeneous base distribution in mitochondrial DNA of Neurospora crassa.

    PubMed Central

    Terpstra, P; Holtrop, M; Kroon, A

    1977-01-01

    The mitochondrial DNA of Neurospora crassa has a heterogeneous intramolecular base distribution. A contiguous piece, representing at least 30% of the total genome, has a G+C content that is 6% lower than the overall G+C content of the DNA. The genes for both ribosomal RNAs are contained in the remaining, relatively G+C rich, part of the genome. PMID:141040

  14. Sensitive determination of DNA based on the interaction between prulifloxacin-terbium(III) complex and DNA.

    PubMed

    Wu, Ting; Fang, Biyun; Chang, Lin; Liu, Min; Chen, Fang

    2013-01-01

    A simple spectrofluorimetric method is described for the determination of DNA, based on its enhancement of the fluorescence intensity of prulifloxacin (PUFX)-Tb(3+). The luminescence intensity of the PUFX-Tb(3+) complex increased up to 10-fold after adding DNA. The excitation and emission wavelengths were 345 and 545 nm, respectively. Under optimum conditions, variations in the fluorescence intensity showed a good linear relationship with the concentration of hsDNA in the range of 3.0 × 10(-9) to 1.0 × 10(-6) g/mL, with a correlation coefficient (R) of 0.997, and the detection limit was 2.1 × 10(-9) g/mL. The method was successfully applied to the determination of DNA in synthetic samples, and recoveries were in the range 97.3-102.0%. The mechanism of fluorescence enhancement of the PUFX-Tb(3+) complex by DNA is also discussed. The mechanism may involve formation of a ternary complex mainly by intercalation binding together with weak electrostatic interaction, which will increase the energy transition from ligand to Tb(3+), increasing the rigidity of the complex, and decreasing the radiationless energy loss through O-H vibration of the H2O molecule in the PUFX-Tb(3+) complex. Compared with the previous DNA probes, the proposed method is not only more robust and friendly to the environment, but also of relatively higher sensitivity.

  15. Outcome of extralevator abdominoperineal excision over conventional abdominoperineal excision for low rectal tumor: a meta-analysis

    PubMed Central

    Yang, Yue; Xu, Huirong; Shang, Zhenhua; Chen, Shouzhen; Chen, Fan; Deng, Qiming; Luo, Li; Zhu, Liang; Shi, Benkang

    2015-01-01

    Objective: A meta-analysis was undertaken to provide an evidence-based basis of clinical trials comparing extralevator abdominoperineal excision with conventional abdominoperineal excision for low rectal tumor. Methods: We searched through the major medical databases such as PubMed, EMBASE, Medline, Science Citation Index, Web of Science for all published studies without any limit on language from January 2009 until January 2015. The following search terms were used: extralevator abdominoperineal excision or cylindrical abdominoperineal resection or conventional abdominoperineal excision or abdominoperineal excision or rectal cancer. Furthermore, Additional related studies were manually searched in the reference lists of all published reviews and retrieved articles. Results: In this meta-analysis, there are a total number of 1797 patients included: 1099 patients in the ELAPE group and 698 in the APE group, and there are not statistically differences between groups in CRM [RR=0.65, 95% CI (0.41, 1.04), P=0.07] and wound complications [RR=1.14, 95% CI (1.09, 1.66), P=0.45] between ELAPE and APE. However, ELAPE has a lower rate of intraoperation perforation [RR=0.44; 95% CI (0.33, 0.60); P<0.00001] and local recurrence [RR=0.45, 95% CI (0.27, 0.77), P=0.003] than APE in terms of short follow-up time. PMID:26628967

  16. Development of DNA-based Identification methods to track the ...

    EPA Pesticide Factsheets

    The ability to track the identity and abundance of larval fish, which are ubiquitous during spawning season, may lead to a greater understanding of fish species distributions in Great Lakes nearshore areas including early-detection of invasive fish species before they become established. However, larval fish are notoriously hard to identify using traditional morphological techniques. While DNA-based identification methods could increase the ability of aquatic resource managers to determine larval fish composition, use of these methods in aquatic surveys is still uncommon and presents many challenges. In response to this need, we have been working with the U. S. Fish and Wildlife Service to develop field and laboratory methods to facilitate the identification of larval fish using DNA-meta-barcoding. In 2012, we initiated a pilot-project to develop a workflow for conducting DNA-based identification, and compared the species composition at sites within the St. Louis River Estuary of Lake Superior using traditional identification versus DNA meta-barcoding. In 2013, we extended this research to conduct DNA-identification of fish larvae collected from multiple nearshore areas of the Great Lakes by the USFWS. The species composition of larval fish generally mirrored that of fish species known from the same areas, but was influenced by the timing and intensity of sampling. Results indicate that DNA-based identification needs only very low levels of biomass to detect pre

  17. Challenges and progress in making DNA-based AIS early ...

    EPA Pesticide Factsheets

    The ability of DNA barcoding to find additional species in hard-to-sample locations or hard-to-identify samples is well established. Nevertheless, adoption of DNA barcoding into regular monitoring programs has been slow, in part due to issues of standardization and interpretation that need resolving. In this presentation, we describe our progress towards incorporating DNA-based identification into broad-spectrum aquatic invasive species early-detection monitoring in the Laurentian Great Lakes. Our work uses community biodiversity information as the basis for evaluating survey performance for various taxonomic groups. Issues we are tackling in bringing DNA-based data to bear on AIS monitoring design include: 1) Standardizing methodology and work flow from field collection and sample handling through bioinformatics post-processing; 2) Determining detection sensitivity and accounting for inter-species differences in DNA amplification and primer affinity; 3) Differentiating sequencing and barcoding errors from legitimate new finds when range and natural history information is limited; and 4) Accounting for the different nature of morphology- vs. DNA-based biodiversity information in subsequent analysis (e.g., via species accumulation curves, multi-metric indices). not applicable

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

    PubMed

    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.

  19. Computational investigation of CNT-based DNA polymerase nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    2015-03-01

    DNA polymerases are important enzymes that replicate DNA molecules with very low error rates - about one error in 105 bases. Recently, it was found that the replication process can be electrically monitored by attaching a Klenow fragment of polymerase I to the surface of a carbon nanotube and monitoring the current along the tube [1]. In this talk, we report results from computational studies on DNA polymerase nanocircuits. We have first performed classical molecular dynamics (MD) calculations to get snapshots of different enzymatic stages, particularly the open state (no DNA binding) and the closed state (DNA double helix binding). We then used density functional theory (DFT) and Keldysh non-equilibrium Green's function (NEGF) formalism to calculate transmission coefficients and currents for each enzymatic state. Our results show that the transmission spectrum and the currents change significantly when the enzyme moves from the open to the closed state. While the initial experiments did not show signal differences between dissimilar bases, the theoretical work in progress is investigating conditions where bases might have distinct signatures, which would allow for DNA sequencing.

  20. Osteophyte excision without cyst excision for a mucous cyst of the finger.

    PubMed

    Lee, H-J; Kim, P-T; Jeon, I-H; Kyung, H-S; Ra, I-H; Kim, T-K

    2014-03-01

    Osteophyte excision is a mainstay of treatment for mucous cyst combined with Heberden's node in a distal interphalangeal joint or in an interphalangeal joint of the thumb. The aim of this study was to evaluate the results of osteophyte excision without cyst excision for the treatment of a mucous cyst combined with Heberden's node. The medical records of 37 patients (42 cases) with a mucous cyst with Heberden's node were retrospectively reviewed. Thirty-eight of 40 cases with available pre-operative simple radiographs showed evidence of joint arthrosis. A T-shaped skin incision of the joint capsule between the extensor tendon and lateral collateral ligament was used. Osteophyte excision without cyst excision was performed. All cysts, except one, regressed without recurrence or a skin complication after osteophyte excision, but eight cases showed post-operative pain and loss of range of motion. Osteophyte excision without cyst excision may be a good treatment choice for mucous cyst of the finger.

  1. Two-base DNA hairpin-loop structures in vivo.

    PubMed Central

    Davison, A; Leach, D R

    1994-01-01

    In vitro studies have revealed that DNA hairpin-loops usually contain four unpaired bases. However, a small subset of sequences can form two-base loops. We have previously described an in vivo assay that is sensitive to tight loop formation and have set out to test whether DNA sequences known to form two-base loops in vitro also form tight loops in vivo. It is shown that the sequences 5'dCNNG and 5'dTNNA behave as predicted if they favour two-base loop formation in vivo, a result that is consistent with previously described in vitro studies. The ability of specific DNA sequences to form tight loops in vivo has implications for their potential to form transient structures involved in gene regulation, recombination and mutagenesis. PMID:7971265

  2. Gold Nanowire Based Electrical DNA Detection Using Rolling Circle Amplification

    PubMed Central

    2014-01-01

    We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from TΩ to kΩ for silver and to Ω for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept. PMID:24433087

  3. DNA nanotechnology based on i-motif structures.

    PubMed

    Dong, Yuanchen; Yang, Zhongqiang; Liu, Dongsheng

    2014-06-17

    CONSPECTUS: Most biological processes happen at the nanometer scale, and understanding the energy transformations and material transportation mechanisms within living organisms has proved challenging. To better understand the secrets of life, researchers have investigated artificial molecular motors and devices over the past decade because such systems can mimic certain biological processes. DNA nanotechnology based on i-motif structures is one system that has played an important role in these investigations. In this Account, we summarize recent advances in functional DNA nanotechnology based on i-motif structures. The i-motif is a DNA quadruplex that occurs as four stretches of cytosine repeat sequences form C·CH(+) base pairs, and their stabilization requires slightly acidic conditions. This unique property has produced the first DNA molecular motor driven by pH changes. The motor is reliable, and studies show that it is capable of millisecond running speeds, comparable to the speed of natural protein motors. With careful design, the output of these types of motors was combined to drive micrometer-sized cantilevers bend. Using established DNA nanostructure assembly and functionalization methods, researchers can easily integrate the motor within other DNA assembled structures and functional units, producing DNA molecular devices with new functions such as suprahydrophobic/suprahydrophilic smart surfaces that switch, intelligent nanopores triggered by pH changes, molecular logic gates, and DNA nanosprings. Recently, researchers have produced motors driven by light and electricity, which have allowed DNA motors to be integrated within silicon-based nanodevices. Moreover, some devices based on i-motif structures have proven useful for investigating processes within living cells. The pH-responsiveness of the i-motif structure also provides a way to control the stepwise assembly of DNA nanostructures. In addition, because of the stability of the i-motif, this

  4. Plasmid DNA-based gene transfer with ultrasound and microbubbles.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Rakugi, Hiromi; Morishita, Ryuichi

    2011-12-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

  5. Monitoring DNA polymerase with nanotube-based nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    DNA polymerases play an important role in the process of life by accurately and efficiently replicating our genetic information. They use a single-stranded DNA as a template and incorporate nucleotides to create the full, double-stranded DNA. Recent experiments have successfully monitored this process by attaching a Klenow fragment of polymerase I to a carbon nanotube and measuring the current along the tube. Follow-up experiments have shown promise for distinguishing between DNA base pairs when nucleotide analogs are used, thus opening a new avenue for DNA sequencing. In this talk, we present results from computational studies on DNA polymerase I nanocircuits. The enzyme was first equilibrated in molecular dynamics and then density functional theory and Keldysh non-equilibrium Green's function methods were used to calculate the ballistic transmission coefficients and currents for different enzymatic states. Our results show significant change in current when the enzyme alternates between open (idle) and closed (synthesizing) states. We can also differentiate between some template bases when modified nucleotides and gate scanning are used.

  6. Base composition at mtDNA boundaries suggests a DNA triple helix model for human mitochondrial DNA large-scale rearrangements.

    PubMed

    Rocher, Christophe; Letellier, Thierry; Copeland, William C; Lestienne, Patrick

    2002-06-01

    Different mechanisms have been proposed to account for mitochondrial DNA (mtDNA) instability based on the presence of short homologous sequences (direct repeats, DR) at the potential boundaries of mtDNA rearrangements. Among them, slippage-mispairing of the replication complex during the asymmetric replication cycle of the mammalian mitochondrial DNA has been proposed to account for the preferential localization of deletions. This mechanism involves a transfer of the replication complex from the first neo-synthesized heavy (H) strand of the DR1, to the DR2, thus bypassing the intervening sequence and producing a deleted molecule. Nevertheless, the nature of the bonds between the DNA strands remains unknown as the forward sequence of DR2, beyond the replication complex, stays double-stranded. Here, we have analyzed the base composition of the DR at the boundaries of mtDNA deletions and duplications and found a skewed pyrimidine content of about 75% in the light-strand DNA template. This suggests the possible building of a DNA triple helix between the G-rich neo-synthesized DR1 and the base-paired homologous G.C-rich DR2. In vitro experiments with the purified human DNA polymerase gamma subunits enabled us to show that the third DNA strand may be used as a primer for DNA replication, using a template with the direct repeat forming a hairpin, with which the primer could initiate DNA replication. These data suggest a novel molecular basis for mitochondrial DNA rearrangements through the distributive nature of the DNA polymerase gamma, at the level of the direct repeats. A general model accounting for large-scale mitochondrial DNA deletion and duplication is proposed. These experiments extend to a DNA polymerase from an eucaryote source the use of a DNA triple helix strand as a primer, like other DNA polymerases from phage and bacterial origins.

  7. Application of DNA-based methods in forensic entomology.

    PubMed

    Wells, Jeffrey D; Stevens, Jamie R

    2008-01-01

    A forensic entomological investigation can benefit from a variety of widely practiced molecular genotyping methods. The most commonly used is DNA-based specimen identification. Other applications include the identification of insect gut contents and the characterization of the population genetic structure of a forensically important insect species. The proper application of these procedures demands that the analyst be technically expert. However, one must also be aware of the extensive list of standards and expectations that many legal systems have developed for forensic DNA analysis. We summarize the DNA techniques that are currently used in, or have been proposed for, forensic entomology and review established genetic analyses from other scientific fields that address questions similar to those in forensic entomology. We describe how accepted standards for forensic DNA practice and method validation are likely to apply to insect evidence used in a death or other forensic entomological investigation.

  8. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles.

    PubMed

    Maier, Alexander M; Weig, Cornelius; Oswald, Peter; Frey, Erwin; Fischer, Peer; Liedl, Tim

    2016-02-10

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials.

  9. Ultrasensitive electrochemical cocaine biosensor based on reversible DNA nanostructure.

    PubMed

    Sheng, Qinglin; Liu, Ruixiao; Zhang, Sai; Zheng, Jianbin

    2014-01-15

    We proposed an ultrasensitive electrochemical cocaine biosensor based on the three-dimensional (3D) DNA structure conversion of nanostructure from Triangular Pyramid Frustum (TPFDNA) to Equilateral Triangle (ETDNA). The presence of cocaine triggered the aptamer-composed DNA nanostructure change from "Close" to "Open", leading to obvious faradaic impedance changes. The unique properties with excellent stability and specific rigid structure of the 3D DNA nanostructure made the biosensing functions stable, sensitive, and regenerable. The Faradaic impedance responses were linearly related to cocaine concentration between 1.0 nM and 2.0 μM with a correlation coefficient of 0.993. The limit of detection was calculated to be 0.21 nM following IUPAC recommendations (3Sb/b). It is expected that the distinctive features of DNA nanostructure would make it potentially advantageous for a broad range of biosensing, bionanoelectronics, and therapeutic applications.

  10. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles

    PubMed Central

    2016-01-01

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  11. Functional Polymorphisms of Base Excision Repair Genes XRCC1 and APEX1 Predict Risk of Radiation Pneumonitis in Patients With Non-Small Cell Lung Cancer Treated With Definitive Radiation Therapy

    SciTech Connect

    Yin Ming; Liao Zhongxing; Liu Zhensheng; Wang, Li-E; Gomez, Daniel; Komaki, Ritsuko; Wei Qingyi

    2011-11-01

    Purpose: To explore whether functional single nucleotide polymorphisms (SNPs) of base-excision repair genes are predictors of radiation treatment-related pneumonitis (RP), we investigated associations between functional SNPs of ADPRT, APEX1, and XRCC1 and RP development. Methods and Materials: We genotyped SNPs of ADPRT (rs1136410 [V762A]), XRCC1 (rs1799782 [R194W], rs25489 [R280H], and rs25487 [Q399R]), and APEX1 (rs1130409 [D148E]) in 165 patients with non-small cell lung cancer (NSCLC) who received definitive chemoradiation therapy. Results were assessed by both Logistic and Cox regression models for RP risk. Kaplan-Meier curves were generated for the cumulative RP probability by the genotypes. Results: We found that SNPs of XRCC1 Q399R and APEX1 D148E each had a significant effect on the development of Grade {>=}2 RP (XRCC1: AA vs. GG, adjusted hazard ratio [HR] = 0.48, 95% confidence interval [CI], 0.24-0.97; APEX1: GG vs. TT, adjusted HR = 3.61, 95% CI, 1.64-7.93) in an allele-dose response manner (Trend tests: p = 0.040 and 0.001, respectively). The number of the combined protective XRCC1 A and APEX1 T alleles (from 0 to 4) also showed a significant trend of predicting RP risk (p = 0.001). Conclusions: SNPs of the base-excision repair genes may be biomarkers for susceptibility to RP. Larger prospective studies are needed to validate our findings.

  12. Antibody-controlled actuation of DNA-based molecular circuits

    PubMed Central

    Engelen, Wouter; Meijer, Lenny H. H.; Somers, Bram; de Greef, Tom F. A.; Merkx, Maarten

    2017-01-01

    DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification. PMID:28211541

  13. Magnetophoretic-based microfluidic device for DNA Concentration.

    PubMed

    Shim, Sangjo; Shim, Jiwook; Taylor, William R; Kosari, Farhad; Vasmatzis, George; Ahlquist, David A; Bashir, Rashid

    2016-04-01

    Nucleic acids serve as biomarkers of disease and it is highly desirable to develop approaches to extract small number of such genomic extracts from human bodily fluids. Magnetic particles-based nucleic acid extraction is widely used for concentration of small amount of samples and is followed by DNA amplification in specific assays. However, approaches to integrate such magnetic particles based capture with micro and nanofluidic based assays are still lacking. In this report, we demonstrate a magnetophoretic-based approach for target-specific DNA extraction and concentration within a microfluidic device. This device features a large chamber for reducing flow velocity and an array of μ-magnets for enhancing magnetic flux density. With this strategy, the device is able to collect up to 95 % of the magnetic particles from the fluidic flow and to concentrate these magnetic particles in a collection region. Then an enzymatic reaction is used to detach the DNA from the magnetic particles within the microfluidic device, making the DNA available for subsequent analysis. Concentrations of over 1000-fold for 90 bp dsDNA molecules is demonstrated. This strategy can bridge the gap between detection of low concentration analytes from clinical samples and a range of micro and nanofluidic sensors and devices including nanopores, nano-cantilevers, and nanowires.

  14. Selection of DNA aptamers with two modified bases

    PubMed Central

    Gawande, Bharat N.; Rohloff, John C.; Carter, Jeffrey D.; von Carlowitz, Ira; Zhang, Chi; Schneider, Daniel J.; Janjic, Nebojsa

    2017-01-01

    The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications. PMID:28265062

  15. Selection of DNA aptamers with two modified bases.

    PubMed

    Gawande, Bharat N; Rohloff, John C; Carter, Jeffrey D; von Carlowitz, Ira; Zhang, Chi; Schneider, Daniel J; Janjic, Nebojsa

    2017-03-14

    The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid-like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

  16. Ab initio Study of Naptho-Homologated DNA Bases

    SciTech Connect

    Sumpter, Bobby G; Vazquez-Mayagoitia, Alvaro; Huertas, Oscar; Fuentes-Cabrera, Miguel A; Orozco, Modesto; Luque, Javier

    2008-01-01

    Naptho-homologated DNA bases have been recently used to build a new type of size expanded DNA known as yyDNA. We have used theoretical techniques to investigate the structure, tautomeric preferences, base-pairing ability, stacking interactions, and HOMO-LUMO gaps of the naptho-bases. The structure of these bases is found to be similar to that of the benzo-fused predecessors (y-bases) with respect to the planarity of the aromatic rings and amino groups. Tautomeric studies reveal that the canonical-like form of naptho-thymine (yyT) and naptho-adenine (yyA) are the most stable tautomers, leading to hydrogen-bonded dimers with the corresponding natural nucleobases that mimic the Watson-Crick pairing. However, the canonical-like species of naptho-guanine (yyG) and naptho-cytosine (yyC) are not the most stable tautomers, and the most favorable hydrogen-bonded dimers involve wobble-like pairings. The expanded size of the naphto-bases leads to stacking interactions notably larger than those found for the natural bases, and they should presumably play a dominant contribution in modulating the structure of yyDNA duplexes. Finally, the HOMO-LUMO gap of the naptho-bases is smaller than that of their benzo-base counterparts, indicating that size-expansion of DNA bases is an efficient way of reducing their HOMO-LUMO gap. These results are examined in light of the available experimental evidence reported for yyT and yyC.

  17. Electron and hole transfer from DNA base radicals to oxidized products of guanine in DNA.

    PubMed

    Cai, Zhongli; Sevilla, Michael D

    2003-03-01

    An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.

  18. Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

    SciTech Connect

    Schormann, Norbert; Zhukovskaya, Natalia; Bedwell, Gregory; Nuth, Manunya; Gillilan, Richard; Prevelige, Peter E.; Ricciardi, Robert P.; Banerjee, Surajit; Chattopadhyay, Debasish

    2016-11-02

    We report that uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. In conclusion, the adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. We provide an overview of the current state of the knowledge on the structure-function relationship of D4.

  19. Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences.

    PubMed

    Wang, Meng; Zhao, Hong-Xia; Wang, Long; Wang, Tao; Yang, Rui-Wu; Wang, Xiao-Li; Zhou, Yong-Hong; Ding, Chun-Bang; Zhang, Li

    2013-10-10

    An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL-F, and psbA-trnH from the chloroplast genome, and ITS, ITS1, and ITS2 from the nuclear genome) and three combinations (rbcL+matK, psbA-trnH+ITS1, and trnL-F+ITS1). The present study collected 232 sequences from 27 Salvia species through DNA sequencing and 77 sequences within the same taxa from the GenBank. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intraspecific and interspecific divergence, DNA barcoding gaps, and identification efficiency via a tree-based method. ITS1 was superior to the other marker for discriminating between species, with an accuracy of 81.48%. The three combinations did not increase species discrimination. Finally, we found that ITS1 is a powerful barcode for identifying Salvia species, especially Salvia miltiorrhiza.

  20. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2014-04-01 2014-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  1. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2012-04-01 2011-04-01 true Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  2. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  3. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  4. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  5. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2011-04-01 2011-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  6. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  7. 27 CFR 70.412 - Excise taxes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 2 2013-04-01 2013-04-01 false Excise taxes. 70.412 Section 70.412 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Beer § 70.412 Excise taxes. (a) Collection. Taxes on distilled spirits, wines, and beer are paid...

  8. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  9. 27 CFR 26.30 - Excise taxes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Excise taxes. 26.30 Section 26.30 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT... Into the United States From Areas Other Than Puerto Rico and the Virgin Islands § 26.30 Excise...

  10. Laparoscopic complete mesocolic excision: West meets East.

    PubMed

    Chow, Carina F K; Kim, Seon Hahn

    2014-10-21

    Complete mesocolic excision is a relatively new concept in western literature. It follows the same concept of total mesorectal excision and units' routinely performing complete mesocolic excisions have good pathological results as well as good improvements in overall survival, disease free survival and local recurrence. And yet unlike total mesorectal excision, uptake in the West has been relatively slow with many units sceptical of the true benefits gained by taking up a more technically challenging and potentially more morbid procedure when there is a paucity of literature to support these claims. This article reviews complete mesocolic excision for colon cancer, attempting to identify the risks and benefits of the technique and particularly looking at the reasons why its uptake has not been universal. It also discusses the similarities of a complete mesocolic excision to a colon resection with a D3 lymphadenectomy as well as the role of a laparoscopic approach to this technique. Considering a D3 lymphadenectomy has been the standard of care for stage II and III colon cancers in many of our Asian neighbours for over 20 years, combining this data with data on complete mesocolic excision may provide enough evidence to support or refute the need for complete mesocolic excisions. Maybe there might be lessons to be learnt from our colleagues in the east.

  11. A MEMS-based miniature DNA analysis system

    SciTech Connect

    Northrup, M.A.; Gonzalez, C.; Hadley, D.

    1995-04-25

    We detail the design and development of a miniature thermal cycling instrument for performing the polymerase chain reaction (PCR) that uses microfabricated, silicon-based reaction chambers. The MEMS-based, battery-operated instrument shows significant improvements over commercial thermal cycling instrumentation. Several different biological systems have been amplified and verified with the miniature PCR instrument including the Human Immunodeficiency Virus; both cloned and genomic DNA templates of {beta} globin; and the genetic disease, Cystic Fibrosis from human DNA. The miniaturization of a PCR thermal cycler is the initial module of a fully-integrated portable, low-power, rapid, and highly efficient bioanalytical instrument.

  12. Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

    PubMed Central

    Rahman, Md. Mahbubur; Li, Xiao-Bo; Lopa, Nasrin Siraj; Ahn, Sang Jung; Lee, Jae-Joon

    2015-01-01

    Conducting polymers (CPs) are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective. PMID:25664436

  13. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  14. Duplex-Selective Ruthenium-based DNA Intercalators

    PubMed Central

    Shade, Chad M.; Kennedy, Robert D.; Rouge, Jessica L.; Rosen, Mari S.; Wang, Mary X.; Seo, Soyoung E.; Clingerman, Daniel J.

    2016-01-01

    We report the design and synthesis of small molecules that exhibit enhanced luminescence in the presence of duplex rather than single-stranded DNA. The local environment presented by a well-known [Ru(dipyrido[2,3-a:3',2'-c]phenazine)L2]2+-based DNA intercalator was modified by functionalizing the bipyridine ligands with esters and carboxylic acids. By systematically varying the number and charge of the pendant groups, it was determined that decreasing the electrostatic interaction between the intercalator and the anionic DNA backbone reduced single-strand interactions and translated to better duplex specificity. In studying this class of complexes, a single RuII complex emerged that selectively luminesces in the presence of duplex DNA with little to no background from interacting with single stranded DNA. This complex shows promise as a new dye capable of selectively staining double versus single-stranded DNA in gel electrophoresis, which cannot be done with conventional SYBR dyes. PMID:26119581

  15. DNA-based species detection capabilities using laser transmission spectroscopy.

    PubMed

    Mahon, A R; Barnes, M A; Li, F; Egan, S P; Tanner, C E; Ruggiero, S T; Feder, J L; Lodge, D M

    2013-01-06

    Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel (Dreissena bugensis) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications.

  16. Flow cytometry-based DNA hybridization and polymorphism analysis

    SciTech Connect

    Cai, H.; Kommander, K.; White, P.S.; Nolan, J.P.

    1998-07-01

    Functional analysis of the humane genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well-suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. The authors are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. The approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advances of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  17. Flow-cytometry-based DNA hybidization and polymorphism analysis

    NASA Astrophysics Data System (ADS)

    Cai, Hong; Kommander, Kristina; White, P. S.; Nolan, John P.

    1998-05-01

    Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  18. Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-Rich DNA, and nuclear DNA analyses

    USGS Publications Warehouse

    Freeman, S.; Pham, M.; Rodriguez, R.J.

    1993-01-01

    Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-rich DNA, and nuclear DNA analyses. Experimental Mycology 17, 309-322. Isolates of Colletotrichum were grouped into 10 separate species based on arbitrarily primed PCR (ap-PCR), A + T-rich DNA (AT-DNA) and nuclear DNA banding patterns. In general, the grouping of Colletotrichum isolates by these molecular approaches corresponded to that done by classical taxonomic identification, however, some exceptions were observed. PCR amplification of genomic DNA using four different primers allowed for reliable differentiation between isolates of the 10 species. HaeIII digestion patterns of AT-DNA also distinguished between species of Colletotrichum by generating species-specific band patterns. In addition, hybridization of the repetitive DNA element (GcpR1) to genomic DNA identified a unique set of Pst 1-digested nuclear DNA fragments in each of the 10 species of Colletotrichum tested. Multiple isolates of C. acutatum, C. coccodes, C. fragariae, C. lindemuthianum, C. magna, C. orbiculare, C. graminicola from maize, and C. graminicola from sorghum showed 86-100% intraspecies similarity based on ap-PCR and AT-DNA analyses. Interspecies similarity determined by ap-PCR and AT-DNA analyses varied between 0 and 33%. Three distinct banding patterns were detected in isolates of C. gloeosporioides from strawberry. Similarly, three different banding patterns were observed among isolates of C. musae from diseased banana.

  19. DNA-based random number generation in security circuitry.

    PubMed

    Gearheart, Christy M; Arazi, Benjamin; Rouchka, Eric C

    2010-06-01

    DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. This research focuses on further developing DNA-based methodologies to mimic digital data manipulation. While exhibiting fundamental principles, this work was done in conjunction with the vision that DNA-based circuitry, when the technology matures, will form the basis for a tamper-proof security module, revolutionizing the meaning and concept of tamper-proofing and possibly preventing it altogether based on accurate scientific observations. A paramount part of such a solution would be self-generation of random numbers. A novel prototype schema employs solid phase synthesis of oligonucleotides for random construction of DNA sequences; temporary storage and retrieval is achieved through plasmid vectors. A discussion of how to evaluate sequence randomness is included, as well as how these techniques are applied to a simulation of the random number generation circuitry. Simulation results show generated sequences successfully pass three selected NIST random number generation tests specified for security applications.

  20. The photoinduced transformation of fluorescent DNA base analogue tC triggers DNA melting.

    PubMed

    Preus, Søren; Jønck, Søren; Pittelkow, Michael; Dierckx, Anke; Karpkird, Thitinun; Albinsson, Bo; Wilhelmsson, L Marcus

    2013-08-01

    While fluorescent analogues of the canonical nucleobases have proven to be highly valuable in a large number of applications, up until today, fluorescent DNA base analogues remain virtually inapplicable for single-molecule fluorescence experiments which require extremely bright and photostable dyes. Insight into the photodegradation processes of these fluorophores is thus a key step in the continuous development towards dyes with improved performances. Here, we show that the commercially available fluorescent nucleobase analogue tC under intense long-term illumination and in the presence of O2 is degraded to form a single photoreaction product which we suggest to be the sulfoxide form of tC. The photoproduct is characterized by a blue-shifted absorption and a less intense fluorescence compared to that of tC. Interestingly, when tC is positioned inside double-stranded DNA this photodriven conversion of tC to its photoproduct greatly reduces the duplex stability of the overall double helix in which the probe is positioned. Since tC can be excited selectively at 400 nm, well outside the absorption band of the natural DNA bases, this observation points towards the application of tC as a general light-triggered switch of DNA duplex stability.

  1. Quantum interference in DNA bases probed by graphene nanoribbons

    NASA Astrophysics Data System (ADS)

    Jeong, Heejeong; Seul Kim, Han; Lee, Sung-Hoon; Lee, Dongho; Hoon Kim, Yong; Huh, Nam

    2013-07-01

    Based on first-principles nonequilibrium Green's function calculations, we demonstrate quantum interference (QI) effects on the tunneling conductance of deoxyribonucleic acid bases placed between zigzag graphene nanoribbon electrodes. With the analogy of QI in hydrocarbon ring structures, we hypothesize that QI can be well preserved in the π-π coupling between the carbon-based electrode and a single DNA base. We demonstrate indications of QI, such as destructively interfered anti-resonance or Fano-resonance, that affect the variation of tunneling conductance depending on the orientation of a base. We find that guanine, with a 10-fold higher transverse conductance, can be singled out from the other bases.

  2. Probe mapping to facilitate transposon-based DNA sequencing

    SciTech Connect

    Strausbaugh, L.D.; Bourke, M.T.; Sommer, M.T.; Coon, M.E.; Berg, C.M. )

    1990-08-01

    A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. The authors demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon {gamma}{delta} (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal {gamma}{delta} segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine {gamma}{delta} insertion sites. The insertions were quire randomly distributed, eve