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Sample records for base excision dna

  1. Modulation of DNA base excision repair during neuronal differentiation

    DEFF Research Database (Denmark)

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

    2013-01-01

    Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize...... DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Wojcik

    2014-10-01

    Full Text Available 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.

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

    NARCIS (Netherlands)

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

    1997-01-01

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER...... and accumulation of DNA base lesions in clinical atherosclerosis is scarce. Here, we evaluated the transcriptional profile of a wide spectrum of BER components as well as DNA damage accumulation in atherosclerotic and non-atherosclerotic arteries. BER gene expression levels were analyzed in 162 carotid plaques, 8...... genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion....

  7. A High Excision Potential of TALENs for Integrated DNA of HIV-Based Lentiviral Vector

    OpenAIRE

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

    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 endonuc...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Hirotaka Ebina

    Full Text Available 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.

  10. Early Steps in the DNA Base Excision Repair Pathway of a Fission Yeast Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Kyoichiro Kanamitsu

    2010-01-01

    Full Text Available DNA base excision repair (BER accounts for maintaining genomic integrity by removing damaged bases that are generated endogenously or induced by genotoxic agents. In this paper, we describe the roles of enzymes functioning in the early steps of BER in fission yeast. Although BER is an evolutionarily conserved process, some unique features of the yeast repair pathway were revealed by genetic and biochemical approaches. AP sites generated by monofunctional DNA glycosylases are incised mainly by AP lyase activity of Nth1p, a sole bifunctional glycosylase in yeast, to leave a blocked 3′ end. The major AP endonuclease Apn2p functions predominantly in removing the 3′ block. Finally, a DNA polymerase fills the gap, and a DNA ligase seals the nick (Nth1p-dependent or short patch BER. Apn1p backs up Apn2p. In long patch BER, Rad2p endonuclease removes flap DNA containing a lesion after DNA synthesis. A UV-specific endonuclease Uve1p engages in an alternative pathway by nicking DNA on the 5′ side of oxidative damage. Nucleotide excision repair and homologous recombination are involved in repair of BER intermediates including the AP site and single-strand break with the 3′ block. Other enzymes working in 3′ end processing are also discussed.

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

    OpenAIRE

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

    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 conside...

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

    DEFF Research Database (Denmark)

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah;

    2015-01-01

    Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function...... of 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....

  13. The new base excision repair pathway in mammals mediated by tyrosyl-DNA-phosphodiesterase 1

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Human tyrosyl-DNA phosphodiesterase 1 (Tdp1 hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety and has been implicated in the repair of Topoisomerase I (TopI-DNA covalent complexes. Tdp1 can also hydrolyze other 3' end DNA alterations including 3' phosphoglycolate and 3' abasic (AP sites, and exhibits the 3' nucleosidase activity indicating that it may function as a general 3' end-processing DNA repair enzyme. Recently we have shown a new Tdp1 activity generating DNA strand break with the 3' phosphate termini from the AP site. AP sites are formed spontaneously and are inevitable intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic, and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair, initiated by DNA glycosylases performing beta, delta-elimination cleavage of the AP sites, has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites that is initiated by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap.

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

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott;

    2014-01-01

    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......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...

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

    Science.gov (United States)

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

    2016-06-21

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

  16. Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Muralidhar L Hegde; Tapas K Hazra; Sankar Mitra

    2008-01-01

    Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3' OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEl, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E.coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3' phosphate ter-mini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APEl. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target.

    Science.gov (United States)

    Fishel, Melissa L; Kelley, Mark R

    2007-01-01

    With our growing understanding of the pathways involved in cell proliferation and signaling, targeted therapies, in the treatment of cancer are entering the clinical arena. New and emerging targets are proteins involved in DNA repair pathways. Inhibition of various proteins in the DNA repair pathways sensitizes cancer cells to DNA damaging agents such as chemotherapy and/or radiation. We study the apurinic endonuclease 1/redox factor-1 (Ape1/Ref-1) and believe that its crucial function in DNA repair and reduction-oxidation or redox signaling make it an excellent target for sensitizing tumor cells to chemotherapy. Ape1/Ref-1 is an essential enzyme in the base excision repair (BER) pathway which is responsible for the repair of DNA caused by oxidative and alkylation damage. As importantly, Ape1/Ref-1 also functions as a redox factor maintaining transcription factors in an active reduced state. Ape1/Ref-1 stimulates the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as AP-1 (Fos/Jun), NFkappaB, HIF-1alpha, CREB, p53 and others. We will discuss what is known regarding the pharmacological targeting of the DNA repair activity, as well as the redox activity of Ape1/Ref-1, and explore the budding clinical utility of inhibition of either of these functions in cancer treatment. A brief discussion of the effect of polymorphisms in its DNA sequence is included because of Ape1/Ref-1's importance to maintenance and integrity of the genome. Experimental modification of Ape1/Ref-1 activity changes the response of cells and of organisms to DNA damaging agents, suggesting that Ape1/Ref-1 may also be a productive target of chemoprevention. In this review, we will provide an overview of Ape1/Ref-1's activities and explore the potential of this protein as a target in cancer treatment as well as its role in chemoprevention.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier, E-mail: didier.gasparutto@cea.fr

    2014-02-17

    Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: 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{sup −1} and 50 μg mL{sup −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

  1. A Review of Recent Experiments on Step-to-Step “Hand-off” of the DNA Intermediates in Mammalian Base Excision Repair Pathways1

    OpenAIRE

    Prasad, R.; Beard, W A; Batra, V. K.; Liu, Y.; Shock, D. D.; Wilson, S H

    2011-01-01

    The current “working model” for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apo...

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

    Science.gov (United States)

    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.

  3. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten

    2010-01-01

    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  4. Isolation of a small molecule inhibitor of DNA base excision repair.

    Science.gov (United States)

    Madhusudan, Srinivasan; Smart, Fiona; Shrimpton, Paul; Parsons, Jason L; Gardiner, Laurence; Houlbrook, Sue; Talbot, Denis C; Hammonds, Timothy; Freemont, Paul A; Sternberg, Michael J E; Dianov, Grigory L; Hickson, Ian D

    2005-01-01

    The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.

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

    Science.gov (United States)

    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.

  6. Effects of post mortem interval and gender in DNA base excision repair activities in rat brains

    Energy Technology Data Exchange (ETDEWEB)

    Soltys, Daniela Tathiana; Pereira, Carolina Parga Martins; Ishibe, Gabriela Naomi; Souza-Pinto, Nadja Cristhina de, E-mail: nadja@iq.usp.br

    2015-06-15

    Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. 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.

    Science.gov (United States)

    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.

  10. DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena K Braithwaite

    Full Text Available Base excision repair (BER is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda, was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

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

    Science.gov (United States)

    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.

  12. Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair

    Science.gov (United States)

    Bulgar, A D; Weeks, L D; Miao, Y; Yang, S; Xu, Y; Guo, C; Markowitz, S; Oleinick, N; Gerson, S L; Liu, L

    2012-01-01

    Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG+/+/UDG−/−). UDG−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG+/+ cells were >10 times more resistant to pemetrexed due to the rapid removal of uracil from DNA by UDG and subsequent repair of the resultant AP sites (abasic sites) via the base excision repair (BER). The resistance to pemetrexed in UDG+/+ cells could be reversed by the addition of methoxyamine (MX), which binds to AP sites and interrupts BER pathway. Furthermore, MX-bound AP sites induced cell death was related to their cytotoxic effect of dual inactivation of UDG and topoisomerase IIα, two genes that are highly expressed in lung cancer cells in comparison with normal cells. Thus, targeting BER-based therapy exhibits more selective cytotoxicity on cancer cells through a synthetic lethal mechanism. PMID:22237209

  13. Base excision repair of oxidative DNA damage and association with cancer and aging

    DEFF Research Database (Denmark)

    Maynard, Scott; Schurman, Shepherd H; Harboe, Charlotte

    2009-01-01

    Aging has been associated with damage accumulation in the genome and with increased cancer incidence. Reactive oxygen species (ROS) are produced from endogenous sources, most notably the oxidative metabolism in the mitochondria, and from exogenous sources, such as ionizing radiation. ROS attack DNA...... readily, generating a variety of DNA lesions, such as oxidized bases and strand breaks. If not properly removed, DNA damage can be potentially devastating to normal cell physiology, leading to mutagenesis and/or cell death, especially in the case of cytotoxic lesions that block the progression of DNA...... recently, BER was shown to also exist in the mitochondria. Here, we review the association of BER of oxidative DNA damage with aging, cancer and other diseases....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-15

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    , it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M......DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA.......T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3-34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89-108]. Protein interactions are not only important for function, but also...

  16. Base excision repair in sugarcane

    Directory of Open Access Journals (Sweden)

    Agnez-Lima Lucymara F.

    2001-01-01

    Full Text Available DNA damage can be induced by a large number of physical and chemical agents from the environment as well as compounds produced by cellular metabolism. This type of damage can interfere with cellular processes such as replication and transcription, resulting in cell death and/or mutations. The low frequency of mutagenesis in cells is due to the presence of enzymatic pathways which repair damaged DNA. Several DNA repair genes (mainly from bacteria, yeasts and mammals have been cloned and their products characterized. The high conservation, especially in eukaryotes, of the majority of genes related to DNA repair argues for their importance in the maintenance of life on earth. In plants, our understanding of DNA repair pathways is still very poor, the first plant repair genes having only been cloned in 1997 and the mechanisms of their products have not yet been characterized. The objective of our data mining work was to identify genes related to the base excision repair (BER pathway, which are present in the database of the Sugarcane Expressed Sequence Tag (SUCEST Project. This search was performed by tblastn program. We identified sugarcane clusters homologous to the majority of BER proteins used in the analysis and a high degree of conservation was observed. The best results were obtained with BER proteins from Arabidopsis thaliana. For some sugarcane BER genes, the presence of more than one form of mRNA is possible, as shown by the occurrence of more than one homologous EST cluster.

  17. A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA

    NARCIS (Netherlands)

    Blainey, Paul C.; Oijen, Antoine M. van; Banerjee, Anirban; Verdine, Gregory L.; Xie, X. Sunney; Hippel, Peter H. von

    2006-01-01

    A central mystery in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding of target sites in DNA. Human oxoguanine DNA glycosylase 1 (hOgg1), for example, must search out rare 8-oxoguanine lesions to prevent transversion mutations arising from o

  18. Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton

    Indian Academy of Sciences (India)

    P Srinivasa Rao; P Sateesh Kumar; Ramesh V Sonti

    2013-03-01

    Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.

  19. The Base Excision Repair system of Salmonella enterica serovar typhimurium counteracts DNA damage by host nitric oxide.

    Directory of Open Access Journals (Sweden)

    Anthony R Richardson

    2009-05-01

    Full Text Available Intracellular pathogens must withstand nitric oxide (NO. generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS and possesses multiple systems to detoxify NO.. Consequently, the level of NO. stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO. sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO. fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO.-dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO.. These observations demonstrate that host-derived NO. damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity.

  20. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Mirta M L Sousa

    Full Text Available Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs. Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ pathway of double-strand break (DSB repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel

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

    NARCIS (Netherlands)

    Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclova, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Diez, Orland; Ramon y Cajal, Teresa; Konstantopoulou, Irene; Martinez-Bouzas, Cristina; Conejero, Raquel Andres; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; 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.; Jonson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herraez, Belen; 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 Joerg; 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; Rodriguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldes, Trinidad; Nevanlinna, Heli; Aittomaki, 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.; Gomez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collee, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; Olah, Edith; Lazaro, Conxi; Teule, Alex; Menendez, 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; 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; Kaulich, Daphne Geschwantler; 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 c

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

    DEFF Research Database (Denmark)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline

    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 th...

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

    DEFF Research Database (Denmark)

    Muftuoglu, Meltem; Kusumoto, Rika; Speina, Elzbieta

    2008-01-01

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

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

    Science.gov (United States)

    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.

  5. Monte carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. II. Comparisons of model predictions to measured data.

    Science.gov (United States)

    Semenenko, V A; Stewart, R D

    2005-08-01

    Clustered damage sites other than double-strand breaks (DSBs) have the potential to contribute to deleterious effects of ionizing radiation, such as cell killing and mutagenesis. In the companion article (Semenenko et al., Radiat. Res. 164, 180-193, 2005), a general Monte Carlo framework to simulate key steps in the base and nucleotide excision repair of DNA damage other than DSBs is proposed. In this article, model predictions are compared to measured data for selected low-and high-LET radiations. The Monte Carlo model reproduces experimental observations for the formation of enzymatic DSBs in Escherichia coli and cells of two Chinese hamster cell lines (V79 and xrs5). Comparisons of model predictions with experimental values for low-LET radiation suggest that an inhibition of DNA backbone incision at the sites of base damage by opposing strand breaks is active over longer distances between the damaged base and the strand break in hamster cells (8 bp) compared to E. coli (3 bp). Model estimates for the induction of point mutations in the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene by ionizing radiation are of the same order of magnitude as the measured mutation frequencies. Trends in the mutation frequency for low- and high-LET radiation are predicted correctly by the model. The agreement between selected experimental data sets and simulation results provides some confidence in postulated mechanisms for excision repair of DNA damage other than DSBs and suggests that the proposed Monte Carlo scheme is useful for predicting repair outcomes.

  6. DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.

    Science.gov (United States)

    Satoh, M S; Jones, C J; Wood, R D; Lindahl, T

    1993-07-01

    Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Remaining closed circular DNA containing other oxygen radical-induced base lesions was used as a substrate for nucleotide excision-repair in a cell-free system. Extracts from normal human cells, but not extracts from xeroderma pigmentosum cells, catalyzed repair synthesis in this DNA. The repair defect in the latter extracts could be specifically corrected by in vitro complementation. The data suggest that accumulation of endogenous oxidative damage in cellular DNA from xeroderma pigmentosum patients contributes to the increased frequency of internal cancers and the neural degeneration occurring in serious cases of the syndrome.

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

    Science.gov (United States)

    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.

  8. Accurate DNA assembly and genome engineering with optimized uracil excision cloning

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna;

    2015-01-01

    Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro......Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway...... that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...

  9. Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?

    Directory of Open Access Journals (Sweden)

    Yi-Chih Tsai

    2013-01-01

    Full Text Available A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP or Hepatitis B virus (HBV tend to give higher levels of oxidative DNA damage (P<0.05. Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P<0.05. Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.

  10. Divergent mechanisms for enzymatic excision of 5-formylcytosine and 5-carboxylcytosine from DNA

    OpenAIRE

    Maiti, Atanu; Michelson, Anna Zhachkina; Armwood, Cherece J; Lee, Jeehiun K.; Drohat, Alexander C.

    2013-01-01

    5-methylcytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), and potentially 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair. Here, we investigate the mechanism for TDG excision of f...

  11. Targeting base excision repair as a sensitization strategy in radiotherapy.

    NARCIS (Netherlands)

    Vens, C.; Begg, A.C.

    2010-01-01

    Cellular DNA repair determines survival after ionizing radiation. Human tumors commonly exhibit aberrant DNA repair since they drive mutagenesis and chromosomal instability. Recent reports have shown alterations in the base excision repair (BER) and single strand break repair (SSBR) pathways in huma

  12. Human ribosomal protein S3 interacts with DNA base excision repair proteins hAPE/Ref-1 and hOGG1.

    Science.gov (United States)

    Hegde, Vijay; Wang, Mu; Deutsch, Walter A

    2004-11-09

    The human ribosomal protein S3 (hS3) possesses associated activities that suggest alternative roles beyond its participation in protein translation. For example, it is capable of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction, an activity that is missing in partially purified extracts of xeroderma pigmentosum group-D fibroblasts. In a recent study, we showed by surface plasmon resonance (SPR) that hS3 also has a very high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) and AP sites in DNA. Using the same SPR technology, it is shown here that hS3 positively interacts with the human base excision repair (BER) enzymes N-glycosylase/AP lyase OGG1 and APE/Ref-1. Using a DNA substrate that allows for the detection of 8-oxoG repair, we also show that hOGG1 N-glycosylase activity becomes increasingly more robust in the presence of hS3. Human S3 was found to co-immunoprecipitate with both hOGG1 and APE/Ref-1, indicating that these proteins physically interact with one another. These results raise the possibility that hS3 not only functions as a ribosomal protein but, in addition, may influence repair activities at sites of DNA damage.

  13. Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

    Directory of Open Access Journals (Sweden)

    Javier Abellón-Ruiz

    2016-01-01

    Full Text Available In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil; Endonuclease V (EndoV, which recognises hypoxanthine; and Endonuclease Q (EndoQ, (which recognises both uracil and hypoxanthine. Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.

  14. Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends

    Energy Technology Data Exchange (ETDEWEB)

    Semenenko, Vladimir; Stewart, Robert D.; Ackerman, Eric J.

    2005-12-31

    Single-cell irradiators and new experimental assays are rapidly expanding our ability to quantify the molecular mechanisms responsible for phenomena such as toxicant-induced adaptations in DNA repair and signal-mediated changes to the genome stability of cells not directly damaged by radiation (i.e., bystander cells). To advance our understanding of, and ability to predict and mitigate, the potentially harmful effects of radiological agents, effective strategies must be devised to incorporate information from molecular and cellular studies into mechanism-based, hierarchical models. A key advantage of the hierarchical modeling approach is that information from DNA repair and other in vitro assays can be systematically integrated into higher-level cell transformation and, eventually, carcinogenesis models. This presentation will outline the hierarchical modeling strategy used to integrate information from in vitro studies into the Virtual Cell (VC) radiobiology software (see Endnote). A new multi-path genomic instability model will be introduced and used to link biochemical processing of double strand breaks (DSBs) to neoplastic cell transformation. Bystander and directly damaged cells are treated explicitly in the model using a microdosimetric approach, although many of the details of the bystander response model are of a necessarily preliminary nature. The new model will be tested against several published radiobiological datasets. Results illustrating how hypothesized bystander mechanisms affect the shape of dose-response curves for neoplastic transformation as a function of Linear Energy Transfer (LET) will be presented. EndNote: R.D. Stewart, Virtual Cell (VC) Radiobiology Software. PNNL-13579, July 2001. Available at http://www.pnl.gov/berc/kbem/vc/ The DNA repair model used in the VC computer program is based on the Two-Lesion Kinetic (TLK) model [Radiat. Res. 156(4), 365-378 October 2001].

  15. Kin-cohort estimates for familial breast cancer risk in relation to variants in DNA base excision repair, BRCA1 interacting and growth factor genes

    Directory of Open Access Journals (Sweden)

    Rutter Joni L

    2004-03-01

    Full Text Available Abstract Background Subtle functional deficiencies in highly conserved DNA repair or growth regulatory processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer. Polymorphisms in DNA repair genes can impact protein function leading to genomic instability facilitated by growth stimulation and increased cancer risk. Thus, 19 single nucleotide polymorphisms (SNPs in eight genes involved in base excision repair (XRCC1, APEX, POLD1, BRCA1 protein interaction (BRIP1, ZNF350, BRCA2, and growth regulation (TGFß1, IGFBP3 were evaluated. Methods Genomic DNA samples were used in Taqman 5'-nuclease assays for most SNPs. Breast cancer risk to ages 50 and 70 were estimated using the kin-cohort method in which genotypes of relatives are inferred based on the known genotype of the index subject and Mendelian inheritance patterns. Family cancer history data was collected from a series of genotyped breast cancer cases (N = 748 identified within a cohort of female US radiologic technologists. Among 2,430 female first-degree relatives of cases, 190 breast cancers were reported. Results Genotypes associated with increased risk were: XRCC1 R194W (WW and RW vs. RR, cumulative risk up to age 70, risk ratio (RR = 2.3; 95% CI 1.3–3.8; XRCC1 R399Q (QQ vs. RR, cumulative risk up to age 70, RR = 1.9; 1.1–3.9; and BRIP1 (or BACH1 P919S (SS vs. PP, cumulative risk up to age 50, RR = 6.9; 1.6–29.3. The risk for those heterozygous for BRCA2 N372H and APEX D148E were significantly lower than risks for homozygotes of either allele, and these were the only two results that remained significant after adjusting for multiple comparisons. No associations with breast cancer were observed for: APEX Q51H; XRCC1 R280H; IGFPB3 -202A>C; TGFß1 L10P, P25R, and T263I; BRCA2 N289H and T1915M; BRIP1 -64A>C; and ZNF350 (or ZBRK1 1845C>T, L66P, R501S, and S472P. Conclusion Some variants in genes within the base-excision repair pathway (XRCC1 and

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ana Osorio

    2014-04-01

    Full Text Available 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.

  19. Developmentally programmed excision of internal DNA sequences in Paramecium aurelia.

    Science.gov (United States)

    Gratias, A; Bétermier, M

    2001-01-01

    The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.

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

    Science.gov (United States)

    2006-11-01

    PROCEDURES2 Materials. The 2′-deoxynucleoside phosphoramidites, CPG supports, and DNA synthesis reagents were purchased from Glen Research (Sterling, VA...flipping enzymes (28). EXPERIMENTAL PROCEDURES Materials. The 2′-deoxynucleoside phosphoramidites, CPG supports, and DNA synthesis reagents were...28425. (21) Klarmann, G. J.; Chen, X.; North, T. W.; Preston, B. D. J. Biol. Chem. 2003, 278, 7902- 7909 . (22) Mansky, L. M.; Preveral, S.; Selig, L

  1. Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Beata Janowska, Marek Komisarski, Paulina Prorok, Beata Sokołowska, Jarosław Kuśmierek, Celina Janion, Barbara Tudek

    2009-01-01

    Full Text Available One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE. HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA- strain were G:C → T:A transversions, occurring within the sequence which in recA+ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.

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

    Directory of Open Access Journals (Sweden)

    Ellen S. Motta

    2010-01-01

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

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

    Science.gov (United States)

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

    1996-10-01

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

  4. DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.

    OpenAIRE

    Satoh, M S; C. J. Jones; Wood, R D; Lindahl, T

    1993-01-01

    Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Rema...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. An HPLC-tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products.

    Science.gov (United States)

    Mullins, Elwood A; Rubinson, Emily H; Pereira, Kevin N; Calcutt, M Wade; Christov, Plamen P; Eichman, Brandt F

    2013-11-01

    DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3 mA) and N7-methylguanine (7 mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described.

  8. Copper-Controllable, Site-Specific DNA Excision in Transgenic Plants

    Institute of Scientific and Technical Information of China (English)

    PENG Xiang-lei; LIANG Bin; CHEN Ming; HU Yuan-lei; LIN Zhong-ping

    2003-01-01

    A copper-inducible, Cre-loxP recombination-mediated DNA excision system has been developed in transgenic tobacco plants. The copper inducible system derived from yeast was used for the control of the expression of the Cre recombinase. Upon copper induction, the GUS reporter gene expression unit flanked by two direct lox sites was excised from the transgenic tobacco genome. Quantitative fluorometric GUS assays,Northern blot and PCR analyses showed a high-efficient, copper-dependent and Cre-loxP mediated DNA recombination in all the tested transgenic lines. The copper inducible foreign gene excision might be of great potential in genetic control of transgenic crops.

  9. Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

    Science.gov (United States)

    Fukuyo, Masaki; Nakano, Toshiaki; Zhang, Yingbiao; Furuta, Yoshikazu; Ishikawa, Ken; Watanabe-Matsui, Miki; Yano, Hirokazu; Hamakawa, Takeshi; Ide, Hiroshi; Kobayashi, Ichizo

    2015-03-11

    The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

  10. X-ray repair cross complementing protein 1 in base excision repair

    DEFF Research Database (Denmark)

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Akbari, Mansour;

    2012-01-01

    X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large...

  11. Ku80-deleted cells are defective at base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Li, Han [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain); Marple, Teresa [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain)

    2013-05-15

    Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H{sub 2}O{sub 2} and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.

  12. DNA repair prognostic index modelling reveals an essential role for base excision repair in influencing clinical outcomes in ER negative and triple negative breast cancers

    Science.gov (United States)

    Abdel-Fatah, Tarek M.A.; Arora, Arvind; Moseley, Paul M.; Perry, Christina; Rakha, Emad A.; Green, Andrew R.; Chan, Stephen Y.T.; Ellis, Ian O.; Madhusudan, Srinivasan

    2015-01-01

    Stratification of oestrogen receptor (ER) negative and triple negative breast cancers (TNBCs) is urgently needed. In the current study, a cohort of 880 ER- (including 635 TNBCs) was immuno-profiled for a panel of DNA repair proteins including: Pol β, FEN1, APE1, XRCC1, SMUG1, PARP1, BRCA1, ATR, ATM, DNA-PKcs, Chk1, Chk2, p53, and TOPO2. Multivariate Cox proportional hazards models (with backward stepwise exclusion of these factors, using a criterion of p < 0.05 for retention of factors in the model) were used to identify factors that were independently associated with clinical outcomes. XRCC1 (p = 0.002), pol β (p = 0.032) FEN1 (p = 0.001) and BRCA1 (p = 0.040) levels were independently associated with poor BCSS. Subsequently, DNA repair index prognostic (DRPI) scores for breast cancer specific survival (BCSS) were calculated and two prognostic groups (DRPI-PGs) were identified. Patients in prognostic group 2 (DRPI-PG2) have higher risk of death (p < 0.001). Furthermore, in DRPI-PG2 patients, exposure to anthracycline reduced the risk of death [(HR (95% CI) = 0.79 (0.64–0.98), p = 0.032) by 21–26%. In addition, DRPI-PG2 patients have adverse clinicopathological features including higher grade, lympho-vascular invasion, Her-2 positive phenotype, compared to those in DRPI-PG1 (p < 0.01). Receiver operating characteristic (ROC) curves indicated that the DRPI outperformed the currently used prognostic factors and adding DRPI to lymph node stage significantly improved their performance as a predictor for BCSS [p < 0.00001, area under curve (AUC) = 0.70]. BER strongly influences pathogenesis of ER- and TNBCs. The DRPI accurately predicts BCSS and can also serve as a valuable prognostic and predictive tool for TNBCs. PMID:26267318

  13. Base excision repair of 8-oxoG in dinucleosomes

    NARCIS (Netherlands)

    H. Menoni (Hervé); M.S. Shukla (Manu Shubhdarshan); V. Gerson (Véronique); S. Dimitrov (Stefan); D. Angelov (Dimitar)

    2012-01-01

    textabstractIn this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particl

  14. Defects in Base Excision Repair Sensitize Cells to Manganese in S. cerevisiae

    Directory of Open Access Journals (Sweden)

    Adrienne P. Stephenson

    2013-01-01

    Full Text Available Manganese (Mn is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn2+ has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn2+-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn2+ were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn2+ caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn2+ as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg2+. These data suggest that Mn2+ causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese.

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

    Science.gov (United States)

    Weng, Liwei; Greenberg, Marc M

    2015-09-01

    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.

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

    Science.gov (United States)

    Charles Richard, John Lalith; Shukla, Manu Shubhdarshan; Menoni, Hervé; Ouararhni, Khalid; Lone, Imtiaz Nisar; Roulland, Yohan; Papin, Christophe; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan

    2016-07-01

    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.

  17. Base excision repair activities differ in human lung cancer cells and corresponding normal controls

    DEFF Research Database (Denmark)

    Karahalil, Bensu; Bohr, Vilhelm A; De Souza-Pinto, Nadja C

    2010-01-01

    Oxidative damage to DNA is thought to play a role in carcinogenesis by causing mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway...... for the repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non......-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three...

  18. Photoreactive DNA as a tool for studying topography of nucleotide excision repair complex

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Nucleotide excision repair (NER is one of the major DNA repair pathways in eukaryotic cells preventing genetic abnormalities caused by DNA damage. NER removes a wide set of structurally diverse lesions such as pyrimidine dimers arising upon UV irradiation and bulky chemical adducts arising upon exposure to environmental carcinogens or chemotherapeutic drugs. In view of the extraordinarily broad substrate specificity of NER, it is of interest to understand how a certain set of proteins recognizes various DNA lesions in the context of a large excess of intact DNA. This review focuses on contribution of photoaffinity labeling technique in the study of DNA damage recognition and following stages resulting in preincision complex assembly, the key and still most unclear steps of NER.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Nicolas Le May

    2010-01-01

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

  1. Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, P.S.; Hevelone, J.; Dwarakanath, V.; Mitchell, D.L. (Texas Christian Univ., Fort Worth (USA))

    1989-06-01

    Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts (cyclobutane dimers and (6-4) photoproducts). Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fonseca, A.S.; Campos, V.M.A.; Magalhaes, L.A.G., E-mail: adnfonseca@ig.com.br [Instituto de Biologia Roberto Alcantara Gomes, Rio de Janeiro, RJ (Brazil). Departamento de Biofisica e Biometria. Lab. de Ciencias Radiologicas; Paoli, F. [Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG (Brazil). Instituto de Ciencias Biologicas. Departamento de Morfologia

    2015-10-15

    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 T{sub 4} endonuclease 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 T{sub 4} endonuclease 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, and iv) did not alter the electrophoretic profile of plasmids incubated with T{sub 4} 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. (author)

  4. Abnormal Base Excision Repair at Trinucleotide Repeats Associated with Diseases: A Tissue-Selective Mechanism

    Directory of Open Access Journals (Sweden)

    Agathi-Vasiliki Goula

    2013-07-01

    Full Text Available More than fifteen genetic diseases, including Huntington’s disease, myotonic dystrophy 1, fragile X syndrome and Friedreich ataxia, are caused by the aberrant expansion of a trinucleotide repeat. The mutation is unstable and further expands in specific cells or tissues with time, which can accelerate disease progression. DNA damage and base excision repair (BER are involved in repeat instability and might contribute to the tissue selectivity of the process. In this review, we will discuss the mechanisms of trinucleotide repeat instability, focusing more specifically on the role of BER.

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

    Science.gov (United States)

    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

  6. Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates

    NARCIS (Netherlands)

    Y. Roche; D. Zhang (Dan); G.M. Segers-Nolten; W. Vermeulen (Wim); C. Wyman (Claire); K. Sugasawa (Kaoru); J.H.J. Hoeijmakers (Jan); C. Otto

    2008-01-01

    textabstractThe interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 9

  7. Replication factor c recruits dna polymerase δ to sites of nucleotide excision repair but is not required for PCNA recruitment

    NARCIS (Netherlands)

    R.M. Overmeer (René); A.M. Gourdin (Audrey); G. Giglia-Mari (Giuseppina); H. Kool (Hanneke); A.B. Houtsmuller (Adriaan); T. Siegal (Tali); M.I. Fousteri (Maria); L.H.F. Mullenders (Leon); W. Vermeulen (Wim)

    2010-01-01

    textabstractNucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincisioncomplexes. 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

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

    Science.gov (United States)

    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. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensa

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

    Science.gov (United States)

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

    Science.gov (United States)

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

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-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.

  12. Mammalian Base Excision Repair: Functional Partnership between PARP-1 and APE1 in AP-Site Repair.

    Directory of Open Access Journals (Sweden)

    Rajendra Prasad

    Full Text Available The apurinic/apyrimidinic- (AP- site in genomic DNA arises through spontaneous base loss and base removal by DNA glycosylases and is considered an abundant DNA lesion in mammalian cells. The base excision repair (BER pathway repairs the AP-site lesion by excising and replacing the site with a normal nucleotide via template directed gap-filling DNA synthesis. The BER pathway is mediated by a specialized group of proteins, some of which can be found in multiprotein complexes in cultured mouse fibroblasts. Using a DNA polymerase (pol β immunoaffinity-capture technique to isolate such a complex, we identified five tightly associated and abundant BER factors in the complex: PARP-1, XRCC1, DNA ligase III, PNKP, and Tdp1. AP endonuclease 1 (APE1, however, was not present. Nevertheless, the complex was capable of BER activity, since repair was initiated by PARP-1's AP lyase strand incision activity. Addition of purified APE1 increased the BER activity of the pol β complex. Surprisingly, the pol β complex stimulated the strand incision activity of APE1. Our results suggested that PARP-1 was responsible for this effect, whereas other proteins in the complex had no effect on APE1 strand incision activity. Studies of purified PARP-1 and APE1 revealed that PARP-1 was able to stimulate APE1 strand incision activity. These results illustrate roles of PARP-1 in BER including a functional partnership with APE1.

  13. The Role of Altered Nucleotide Excision Repair and UVB-Induced DNA Damage in Melanomagenesis

    Directory of Open Access Journals (Sweden)

    Timothy Budden

    2013-01-01

    Full Text Available UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V. XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis.

  14. Initial steps of the base excision repair pathway within the nuclear architecture; Les etapes initiales du mecanisme de reparation par excision de bases au sein de l'architecture nucleaire

    Energy Technology Data Exchange (ETDEWEB)

    Amouroux, R

    2009-09-15

    Oxidative stress induced lesions threaten aerobic organisms by representing a major cause of genomic instability. A common product of guanine oxidation, 8-oxo-guanine (8- oxoG) is particularly mutagenic by provoking G to T transversions. Removal of oxidised bases from DNA is initiated by the recognition and excision of the damaged base by a DNA glycosylase, initiating the base excision repair (BER) pathway. In mammals, 8-oxoG is processed by the 8-oxoG-DNA-glycosylase I (OGG1), which biochemical mechanisms has been well characterised in vitro. However how and where this enzyme finds the modified base within the complex chromatin architecture is not yet understood. We show that upon induction of 8-oxoG, OGG1, together with at least two other proteins involved in BER, is recruited from a soluble fraction to chromatin. Formation kinetics of this patches correlates with 8-oxoG excision, suggesting a direct link between presence of this chromatin-associated complexes and 8-oxoG repair. More precisely, these repair patches are specifically directed to euchromatin regions, and completely excluded from heterochromatin regions. Inducing of artificial chromatin compaction results in a complete inhibition of the in vivo repair of 8-oxoG, probably by impeding the access of OGG1 to the lesion. Using OGG1 mutants, we show that OGG1 direct recognition of 8-oxoG did not trigger its re-localisation to the chromatin. We conclude that in response to the induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions. (author)

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

    DEFF Research Database (Denmark)

    Skarpengland, Tonje; Holm, Sverre; Scheffler, Katja

    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 cl...... 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....... 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...

  16. Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae.

    Science.gov (United States)

    Czaja, Wioletta; Mao, Peng; Smerdon, Michael J

    2014-04-01

    The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death.

    Science.gov (United States)

    Bissenbaev, Amangeldy K; Ishchenko, Alexander A; Taipakova, Sabira M; Saparbaev, Murat K

    2011-10-01

    Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the β-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

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

    Directory of Open Access Journals (Sweden)

    Ankita Shukla

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong-Min; Choi, Ji Ye [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Yi, Joo Mi [Research Center, Dongnam Institute of Radiological & Medical Sciences, Busan (Korea, Republic of); Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Kang, Tae-Hong, E-mail: thkang@dau.ac.kr [Department of Biological Science, Dong-A University, Busan (Korea, Republic of)

    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.

  2. Detoxification of olefinic epoxides and nucleotide excision repair of epoxide-mediated DNA damage: Insights from animal models examining human sensitivity to 1,3-butadiene.

    Science.gov (United States)

    Wickliffe, Jeffrey K; Herring, Stacy M; Hallberg, Lance M; Galbert, Lori A; Masters, Oscar E; Ammenheuser, Marinel M; Xie, Jingwu; Friedberg, Errol C; Lloyd, R Stephen; Abdel-Rahman, Sherif Z; Ward, Jonathan B

    2007-03-20

    1,3-Butadiene (BD) is a well-documented mutagen and carcinogen in rodents and is currently classified as a probable carcinogen in humans. Studies investigating workers exposed to BD indicate that, in some plants, there may be an increased genetic risk, and that polymorphisms in biotransformation and DNA repair proteins may modulate genetic susceptibility. To investigate the role of genetic polymorphisms in microsomal epoxide hydrolase (mEH) or nucleotide excision repair (NER) in contributing to the mutagenicity of BD, we conducted a series of experiments in which mice lacking mEH or NER activity were exposed to BD by inhalation or to the reactive epoxide metabolites of BD (epoxybutene-EB or diepoxybutane-DEB) by i.p. injection. Genetic susceptibility was measured using the Hprt cloning assay. Both deficient strains of mouse were significantly more sensitive to the mutagenic effects of BD and the injected epoxides. These studies provide support for the critical role that mEH plays in the biotransformation of BD, and the role that NER plays in maintaining genomic integrity following exposure to BD. Additional studies are needed to examine the importance of base excision repair (BER) in maintaining genomic integrity, the differential formation of DNA and protein adducts in deficient strains, and the potential for enhanced sensitivity to BD genotoxicity in mice either lacking or deficient in both biotransformation and DNA repair activity.

  3. 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.

    Science.gov (United States)

    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.

  4. Alzheimer’s Disease-Associated Neurotoxic Peptide Amyloid-Β Impairs Base Excision Repair in Human Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Anne Forestier

    2012-11-01

    Full Text Available Alzheimer’s disease (AD is the leading cause of dementia in developed countries. It is characterized by two major pathological hallmarks, one of which is the extracellular aggregation of the neurotoxic peptide amyloid-β (Aβ, which is known to generate oxidative stress. In this study, we showed that the presence of Aβ in a neuroblastoma cell line led to an increase in both nuclear and mitochondrial DNA damage. Unexpectedly, a concomitant decrease in basal level of base excision repair, a major route for repairing oxidative DNA damage, was observed at the levels of both gene expression and protein activity. Moreover, the addition of copper sulfate or hydrogen peroxide, used to mimic the oxidative stress observed in AD-affected brains, potentiates Aβ-mediated perturbation of DNA damage/repair systems in the “Aβ cell line”. Taken together, these findings indicate that Aβ could act as double-edged sword by both increasing oxidative nuclear/mitochondrial damage and preventing its repair. The synergistic effects of increased ROS production, accumulated DNA damage and impaired DNA repair could participate in, and partly explain, the massive loss of neurons observed in Alzheimer’s disease since both oxidative stress and DNA damage can trigger apoptosis.

  5. Base of tongue neurilemmoma: excision by transoral laser microsurgery.

    Science.gov (United States)

    Ballesteros, Ferran; Vilaseca, Isabel; Blanch, Jose Luis; Gaspa, Albert; Bernal-Sprekelsen, Manuel

    2007-09-01

    Benign and malignant tumors of the tongue base can be removed by various surgical approaches. A rare case of neurilemmoma of the base of the tongue removed by transoral laser microsurgery (TLM) is presented. Schwannomas in this area usually present as a slow-growing, painless mass on the tongue surface. In this case, fiberoptic examination and magnetic resonance imaging were crucial as first step studies to elucidate the biology of the lesion.

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

    Science.gov (United States)

    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 ...

  7. Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-induced DNA Damage via Physical Interaction with Xeroderma Pigmentosum Group A*

    Science.gov (United States)

    Shell, Steven M.; Li, Zhengke; Shkriabai, Nikolozi; Kvaratskhelia, Mamuka; Brosey, Chris; Serrano, Moises A.; Chazin, Walter J.; Musich, Phillip R.; Zou, Yue

    2009-01-01

    In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation. PMID:19586908

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

    Science.gov (United States)

    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.

  9. Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer's disease.

    Science.gov (United States)

    Gredilla, Ricardo; Weissman, Lior; Yang, Jenq-Lin; Bohr, Vilhelm A; Stevnsner, Tinna

    2012-04-01

    Brain aging is associated with synaptic decline and synaptic function is highly dependent on mitochondria. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of several neurodegenerative diseases. Here we have investigated the repair of oxidative base damage, in synaptosomes of mouse brain during normal aging and in an AD model. During normal aging, a reduction in the base excision repair (BER) capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of presymptomatic and symptomatic AD mice harboring mutated amyolid precursor protein (APP), Tau, and presinilin-1 (PS1) (3xTgAD). Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed.

  10. UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kelley N. Newton

    2012-01-01

    Full Text Available UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  12. Is There a Role for Base Excision Repair in Estrogen/Estrogen Receptor-Driven Breast Cancers?

    Science.gov (United States)

    Abdel-Fatah, Tarek M.A.; Perry, Christina; Arora, Arvind; Thompson, Nicola; Doherty, Rachel; Moseley, Paul M.; Green, Andrew R.; Chan, Stephen Y.T.; Ellis, Ian O.

    2014-01-01

    Abstract Estrogen and estrogen metabolite-induced reactive oxygen species generation can promote oxidative DNA base damage. If unrepaired, base damaging lesions could accelerate mutagenesis, leading to a “mutator phenotype” characterized by aggressive behavior in estrogen-estrogen receptor (ER)-driven breast cancer. To test this hypothesis, we investigated 1406 ER+ early-stage breast cancers with 20 years' long-term clinical follow-up data for DNA polymerase β (pol β), flap endonuclease 1 (FEN1), AP endonuclease 1 (APE1), X-ray cross-complementation group 1 protein (XRCC1), single-strand monofunctional uracil glycosylase-1 (SMUG1), poly (ADP-ribose) polymerase 1 (PARP1), ataxia telangiectasia mutated and Rad3 related (ATR), ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Chk1, Chk2, p53, breast cancer susceptibility gene 1 (BRCA1), and topoisomerase 2 (TOPO2) expression. Multivariate Cox proportional hazards model was used to calculate a DNA repair prognostic index and correlated to clinicopathological variables and survival outcomes. Key base excision repair (BER) proteins, including XRCC1, APE1, SMUG1, and FEN1, were independently associated with poor breast cancer-specific survival (BCSS) (ps≤0.01). Multivariate Cox model stratified patients into four distinct prognostic sub-groups with worsening BCSS (ps<0.01). In addition, compared with prognostic sub-group 1, sub-groups 2, 3, and 4 manifest increasing tumor size, grade, mitosis, pleomorphism, differentiation, lymphovascular invasion, high Ki67, loss of Bcl-2, luminal B phenotype (ps≤0.01), and poor survival, including in patients who received tamoxifen adjuvant therapy (p<0.00001). Our observation supports the hypothesis that BER-directed stratification could inform appropriate therapies in estrogen-ER-driven breast cancers. Antioxid. Redox Signal. 21, 2262–2268. PMID:25111287

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

    Science.gov (United States)

    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

  14. Removal of the bloom syndrome DNA helicase extends the utility of imprecise transposon excision for making null mutations in Drosophila.

    Science.gov (United States)

    Witsell, Alice; Kane, Daniel P; Rubin, Sarah; McVey, Mitch

    2009-11-01

    Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements.

  15. Haploinsufficiency for BRCA1 is associated with normal levels of DNA nucleotide excision repair in breast tissue and blood lymphocytes

    Directory of Open Access Journals (Sweden)

    Johnson Jennifer M

    2005-06-01

    Full Text Available Abstract Background Screening mammography has had a positive impact on breast cancer mortality but cannot detect all breast tumors. In a small study, we confirmed that low power magnetic resonance imaging (MRI could identify mammographically undetectable tumors by applying it to a high risk population. Tumors detected by this new technology could have unique etiologies and/or presentations, and may represent an increasing proportion of clinical practice as new screening methods are validated and applied. A very important aspect of this etiology is genomic instability, which is associated with the loss of activity of the breast cancer-predisposing genes BRCA1 and BRCA2. In sporadic breast cancer, however, there is evidence for the involvement of a different pathway of DNA repair, nucleotide excision repair (NER, which remediates lesions that cause a distortion of the DNA helix, including DNA cross-links. Case presentation We describe a breast cancer patient with a mammographically undetectable stage I tumor identified in our MRI screening study. She was originally considered to be at high risk due to the familial occurrence of breast and other types of cancer, and after diagnosis was confirmed as a carrier of a Q1200X mutation in the BRCA1 gene. In vitro analysis of her normal breast tissue showed no differences in growth rate or differentiation potential from disease-free controls. Analysis of cultured blood lymphocyte and breast epithelial cell samples with the unscheduled DNA synthesis (UDS assay revealed no deficiency in NER. Conclusion As new breast cancer screening methods become available and cost effective, patients such as this one will constitute an increasing proportion of the incident population, so it is important to determine whether they differ from current patients in any clinically important ways. Despite her status as a BRCA1 mutation carrier, and her mammographically dense breast tissue, we did not find increased cell

  16. SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner

    Science.gov (United States)

    Xu, Zhu; Zhang, Lei; Zhang, Wenjun; Meng, Du; Zhang, Hongxia; Jiang, Ying; Xu, Xiaojun; Van Meter, Michael; Seluanov, Andrei; Gorbunova, Vera; Mao, Zhiyong

    2015-01-01

    In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER. PMID:25607651

  17. DNA Based Molecular Scale Nanofabrication

    Science.gov (United States)

    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...addition, we have expanded our efforts to include DNA- mediated HF etching of SiÜ2, DNA- mediated nanoimprinting lithography, DNA-based patterning of self...detailed kinetics study of DNA- mediated chemical reactions. Examples of such reactions include chemical vapor deposition (CVD) of inorganic oxide and HF

  18. Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.

    Science.gov (United States)

    Mirbahai, Leda; Kershaw, Rachael M; Green, Richard M; Hayden, Rachel E; Meldrum, Rosalind A; Hodges, Nikolas J

    2010-02-01

    An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.

  19. Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair

    NARCIS (Netherlands)

    A. Campalans (Anna); R. Amouroux (Rachel); H. Menoni (Hervé); W. Vermeulen (Wim); J.P. Radicella (Pablo)

    2013-01-01

    textabstractSingle-strand break repair (SSBR) and base excision repair (BER) of modified bases and abasic sites share several players. Among them is XRCC1, an essential scaffold protein with no enzymatic activity, required for the coordination of both pathways. XRCC1 is recruited to SSBR by PARP-1,

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. The role of base excision repair in the development of primary open angle glaucoma in the Polish population

    Energy Technology Data Exchange (ETDEWEB)

    Cuchra, Magda; Markiewicz, Lukasz; Mucha, Bartosz [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz (Poland); Pytel, Dariusz [The Abramson Family Cancer Research Institute, Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425 (United States); Szymanek, Katarzyna [Department of Ophthalmology, Medical University of Warsaw, SPKSO Hospital, Warsaw (Poland); Szemraj, Janusz [Department of Medical Biochemistry, Medical University of Lodz, Lodz (Poland); Szaflik, Jerzy; Szaflik, Jacek P. [Department of Ophthalmology, Medical University of Warsaw, SPKSO Hospital, Warsaw (Poland); Majsterek, Ireneusz, E-mail: ireneusz.majsterek@umed.lodz.pl [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz (Poland)

    2015-08-15

    Highlights: • We suggested the association of XRCC1 gene with the increase risk of POAG development. • We indicated the association of clinical factor and XRCC1, MUTYH, ADPRT and APE1 genes with POAG progression. • We postulated the increase level of oxidative DNA damage in group of patients with POAG in relation to healthy controls. • We suggested the slightly decrease ability to repair of oxidative DNA damage. • This is the first data that showed the role of BER mechanism in POAG pathogenesis. - Abstract: Glaucoma is a leading cause of irreversible blindness in developing countries. Previous data have shown that progressive loss of human TM cells may be connected with chronic exposure to oxidative stress. This hypothesis may suggest a role of the base excision repair (BER) pathway of oxidative DNA damage in primary open angle glaucoma (POAG) patients. The aim of our study was to evaluate an association of BER gene polymorphism with a risk of POAG. Moreover, an association of clinical parameters was examined including cup disk ratio (c/d), rim area (RA) and retinal nerve fiber layer (RNFL) with glaucoma progression according to BER gene polymorphisms. Our research included 412 patients with POAG and 454 healthy controls. Gene polymorphisms were analyzed by PCR-RFLP. Heidelberg Retinal Tomography (HRT) clinical parameters were also analyzed. The 399Arg/Gln genotype of the XRCC1 gene (OR 1.38; 95% CI 1.02–1.89 p = 0.03) was associated with an increased risk of POAG occurrence. It was indicated that the 399Gln/Gln XRCC1 genotype might increase the risk of POAG progression according to the c/d ratio (OR 1.67; 95% CI 1.07–2.61 P = 0.02) clinical parameter. Moreover, the association of VF factor with 148Asp/Glu of APE1 genotype distribution and POAG progression (OR 2.25; 95% CI 1.30–3.89) was also found. Additionally, the analysis of the 324Gln/His MUTYH polymorphism gene distribution in the patient group according to RNFL factor showed that it might

  3. Quantum mechanical study of the β- and δ-lyase reactions during the base excision repair process: application to FPG.

    Science.gov (United States)

    Sowlati-Hashjin, Shahin; Wetmore, Stacey D

    2015-10-14

    Bacterial FPG (or MutM) is a bifunctional DNA glycosylase that is primarily responsible for excising 8-oxoguanine (OG) from the genome by cleaving the glycosidic bond and the DNA backbone at the 3'- and 5'-phosphates of the damaged nucleoside. In the present work, quantum mechanical methods (SMD-M06-2X/6-311+G(2df,2p)//IEF-PCM-B3LYP/6-31G(d)) and a ring-opened Schiff base model that includes both the 3'- and 5'-phosphate groups are used to investigate the β- and δ-elimination reactions facilitated by FPG. Both the β- and δ-elimination reactions are shown to proceed through an E1cB mechanism that involves proton abstraction prior to the phosphate-ribose bond cleavage. Since transition states for the phosphate elimination reactions could not be characterized in the absence of leaving group protonation, our work confirms that the phosphate elimination reactions require protonation by a residue in the FPG active site, and can likely be further activated by additional active-site interactions. Furthermore, our model suggests that 5'-PO4 activation may proceed through a nearly isoenergetic direct (intramolecular) proton transfer involving the O4' proton of the deoxyribose of the damaged nucleoside. Regardless, our model predicts that both 3'- and 5'-phosphate protonation and elimination steps occur in a concerted reaction. Most importantly, our calculated barriers for the phosphate cleavage reactions reveal inherent differences between the β- and δ-elimination steps. Indeed, our calculations provide a plausible explanation for why the δ-elimination rather than the β-elimination is the rate-determining step in the BER facilitated by FPG, and why some bifunctional glycosylases (including the human counterpart, hOgg1) lack δ-lyase activity. Together, the new mechanistic features revealed by our work can be used in future large-scale modeling of the DNA-protein system to unveil the roles of key active sites residues in these relatively unexplored BER steps.

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

    Science.gov (United States)

    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 laser, but increase in muscle tissue (p  0.05), but ERCC2 mRNA expression decreases in skin (p 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.

  5. The role of base excision repair in the development of primary open angle glaucoma in the Polish population.

    Science.gov (United States)

    Cuchra, Magda; Markiewicz, Lukasz; Mucha, Bartosz; Pytel, Dariusz; Szymanek, Katarzyna; Szemraj, Janusz; Szaflik, Jerzy; Szaflik, Jacek P; Majsterek, Ireneusz

    2015-08-01

    Glaucoma is a leading cause of irreversible blindness in developing countries. Previous data have shown that progressive loss of human TM cells may be connected with chronic exposure to oxidative stress. This hypothesis may suggest a role of the base excision repair (BER) pathway of oxidative DNA damage in primary open angle glaucoma (POAG) patients. The aim of our study was to evaluate an association of BER gene polymorphism with a risk of POAG. Moreover, an association of clinical parameters was examined including cup disk ratio (c/d), rim area (RA) and retinal nerve fiber layer (RNFL) with glaucoma progression according to BER gene polymorphisms. Our research included 412 patients with POAG and 454 healthy controls. Gene polymorphisms were analyzed by PCR-RFLP. Heidelberg Retinal Tomography (HRT) clinical parameters were also analyzed. The 399 Arg/Gln genotype of the XRCC1 gene (OR 1.38; 95% CI 1.02-1.89 p = 0.03) was associated with an increased risk of POAG occurrence. It was indicated that the 399 Gln/Gln XRCC1 genotype might increase the risk of POAG progression according to the c/d ratio (OR 1.67; 95% CI 1.07-2.61 P = 0.02) clinical parameter. Moreover, the association of VF factor with 148 Asp/Glu of APE1 genotype distribution and POAG progression (OR 2.25; 95% CI 1.30-3.89) was also found. Additionally, the analysis of the 324 Gln/His MUTYH polymorphism gene distribution in the patient group according to RNFL factor showed that it might decrease the progression of POAG (OR 0.47; 95% CI 0.30-0.82 P = 0.005). We suggest that the 399 Arg/Gln polymorphism of the XRCC1 gene may serve as a predictive risk factor of POAG.

  6. Accurate Dna Assembly And Direct Genome Integration With Optimized Uracil Excision Cloning To Facilitate Engineering Of Escherichia Coli As A Cell Factory

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Kim, Se Hyeuk; Nørholm, Morten

    2015-01-01

    Plants produce a vast diversity of valuable compounds with medical properties, but these are often difficult to purify from the natural source or produce by organic synthesis. An alternative is to transfer the biosynthetic pathways to an efficient production host like the bacterium Escherichia co......-excision-based cloning and combining it with a genome-engineering approach to allow direct integration of whole metabolic pathways into the genome of E. coli, to facilitate the advanced engineering of cell factories....

  7. The mitochondrial transcription factor A functions in mitochondrial base excision repair

    DEFF Research Database (Denmark)

    Canugovi, Chandrika; Maynard, Scott; Bayne, Anne-Cécile V

    2010-01-01

    Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids. TFAM plays an important role in mitochondrial transcription and replication. TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected i...

  8. Cockayne syndrome: varied requirement of transcription-coupled nucleotide excision repair for the removal of three structurally different adducts from transcribed DNA.

    Directory of Open Access Journals (Sweden)

    Nataliya Kitsera

    Full Text Available Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS, however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N2-yl-2-acetylaminofluorene adduct (dG(N2-AAF constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is removed exclusively by the CSA- and CSB-dependent pathway. In contrast, contribution of the CS proteins to the removal of two other transcription-blocking DNA lesions - N-(deoxyguanosin-8-yl-2-acetylaminofluorene (dG(C8-AAF and cyclobutane thymine-thymine (TT dimer - is only minor (TT dimer or none (dG(C8-AAF. The unique properties of dG(N2-AAF identify this adduct as a prototype for a new class of DNA lesions that escape the alternative global genome repair and could be critical for the CS pathogenesis.

  9. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  10. H. pylori-Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression

    Directory of Open Access Journals (Sweden)

    Mara L. Hartung

    2015-10-01

    Full Text Available The human bacterial pathogen Helicobacter pylori exhibits genotoxic properties that promote gastric carcinogenesis. H. pylori introduces DNA double strand breaks (DSBs in epithelial cells that trigger host cell DNA repair efforts. Here, we show that H. pylori-induced DSBs are repaired via error-prone, potentially mutagenic non-homologous end-joining. A genome-wide screen for factors contributing to DSB induction revealed a critical role for the H. pylori type IV secretion system (T4SS. Inhibition of transcription, as well as NF-κB/RelA-specific RNAi, abrogates DSB formation. DSB induction further requires β1-integrin signaling. DSBs are introduced by the nucleotide excision repair endonucleases XPF and XPG, which, together with RelA, are recruited to chromatin in a highly coordinated, T4SS-dependent manner. Interestingly, XPF/XPG-mediated DNA DSBs promote NF-κB target gene transactivation and host cell survival. In summary, H. pylori induces XPF/XPG-mediated DNA damage through activation of the T4SS/β1-integrin signaling axis, which promotes NF-κB target gene expression and host cell survival.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

    2013-06-15

    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 eight DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by32P-postlabeling. Multivariable regression models were compared by Akaike's information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 10(8) 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 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 three to four 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.

  13. DNA-based hybrid catalysis.

    Science.gov (United States)

    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.

  14. Efficient simultaneous excision of multiple selectable marker cassettes using I-SceI-induced double-strand DNA breaks in Saccharomyces cerevisiae.

    Science.gov (United States)

    Solis-Escalante, Daniel; Kuijpers, Niels G A; van der Linden, Franka H; Pronk, Jack T; Daran, Jean-Marc; Daran-Lapujade, Pascale

    2014-08-01

    Large strain construction programs and functional analysis studies are becoming commonplace in Saccharomyces cerevisiae and involve construction of strains that carry multiple selectable marker genes. Extensive strain engineering is, however, severely hampered by the limited number of recyclable marker genes and by the reduced genome stability that occurs upon repeated use of heterologous recombinase-based marker removal methods. The present study proposes an efficient method to recycle multiple markers in S. cerevisiae simultaneously, thereby circumventing shortcomings of existing techniques and substantially accelerating the process of selection-excision. This method relies on artificial generation of double-strand breaks around the selection marker cassette by the meganuclease I-SceI and the subsequent repair of these breaks by the yeast homologous recombination machinery, guided by direct repeats. Simultaneous removal of up to three marker cassettes was achieved with high efficiencies (up to 56%), suggesting that I-SceI-based marker removal has the potential to co-excise an even larger number of markers. This locus- and marker-independent method can be used for both dominant and auxotrophy-complementing marker genes. Seven pDS plasmids carrying various selectable markers, which can be used for PCR-based generation of deletion cassettes suited for I-SceI marker recycling, are described and made available to the scientific community.

  15. Investigations on the role of base excision repair and non-homologous end-joining pathways in sodium selenite-induced toxicity and mutagenicity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Mániková, Dominika; Vlasáková, Danusa; Loduhová, Jana; Letavayová, Lucia; Vigasová, Dana; Krascsenitsová, Eva; Vlcková, Viera; Brozmanová, Jela; Chovanec, Miroslav

    2010-03-01

    Selenium (Se) belongs to nutrients that are essential for human health. Biological activity of this compound, however, mainly depends on its dose, with a potential of Se to induce detrimental effects at high doses. Although mechanisms lying behind detrimental effects of Se are poorly understood yet, they involve DNA damage induction. Consequently, DNA damage response and repair pathways may play a crucial role in cellular response to Se. Using Saccharomyces cerevisiae we showed that sodium selenite (SeL), an inorganic form of Se, can be toxic and mutagenic in this organism due to its ability to induce DNA double-strand breaks (DSBs). Moreover, we reported that a spectrum of mutations induced by this compound in the stationary phase of growth is mainly represented by 1-4 bp deletions. Consequently, we proposed that SeL acts as an oxidizing agent in yeast producing oxidative damage to DNA. As short deletions could be anticipated to arise as a result of action of non-homologous end-joining (NHEJ) and oxidative damage to DNA is primarily coped with base excision repair (BER), a contribution of these two pathways towards survival, DSB induction, mutation frequency and types of mutations following SeL exposure was examined in present study. First, we show that while NHEJ plays no role in repairing toxic DNA lesions induced by SeL, cells with impairment in BER are sensitized towards this compound. Of BER activities examined, those responsible for processing of 3'-blocking DNA termini seem to be the most crucial for manifestation of the toxic effects of SeL in yeast. Second, an impact of NHEJ and BER on DSB induction after SeL exposure turned to be inappreciable, as no increase in DNA double-strand breakage in NHEJ and BER single or NHEJ BER double mutant upon SeL exposure was observed. Finally, we demonstrate that impairment in both these pathways does not importantly change mutation frequency after SeL exposure and that NHEJ is not responsible for generation of short

  16. DNA fragments assembly based on nicking enzyme system.

    Directory of Open Access Journals (Sweden)

    Rui-Yan Wang

    Full Text Available 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.

  17. Incorporation of dUMP into DNA is a major source of spontaneous DNA damage, while excision of uracil is not required for cytotoxicity of fluoropyrimidines in mouse embryonic fibroblasts.

    Science.gov (United States)

    Andersen, Sonja; Heine, Tina; Sneve, Ragnhild; König, Imbritt; Krokan, Hans E; Epe, Bernd; Nilsen, Hilde

    2005-03-01

    Uracil may arise in DNA as a result of deamination of cytosine or through incorporation of dUMP instead of dTMP during replication. We have studied the steady-state levels of uracil in the DNA of primary cells and mouse embryonic fibroblast (MEF) cell lines from mice deficient in the Ung uracil-DNA glycosylase. The results show that the levels of uracil in the DNA of Ung(-/-) cells strongly depend on proliferation, indicating that the uracil residues originate predominantly from misincorporation during replication. Treatment with 5-fluoro-2'-deoxyuridine (5-FdUrd) or 5-fluorouracil (5-FU) gives rise to a dose-dependent increase of uracil in Ung(-/-) MEFs (up to 1.5-fold) but not in wild-type cells. Interestingly, Ung(-/-) MEFs accumulate AP-sites as well as uracil in response to 5-FdUrd but not to 5-FU. This accumulation of repair intermediates suggests a loss of tightly co-ordinated repair in the absence of Ung, and correlates with stronger inhibition of cell proliferation in response to 5-FdUrd, but not to 5-FU, in Ung(-/-) MEFs compared with wild-type cells. However, other cytotoxic effects of these fluoropyrimidines are comparable in both wild-type and Ung-deficient cells, demonstrating that excision of uracil from DNA by the Ung uracil-DNA glycosylase is not a prerequisite for obtaining cytotoxicity.

  18. REPAIR OF LARGE SKULL BASE DEFECT FOLLOWING EXCISION OF BASALOID SQUAMOUS CELL CARCINOMA OF MAXILLO - ETHMOID REGION : A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Monoj Mukherjee

    2015-02-01

    Full Text Available AIM: To present a case of basaloid squamous cell carcinoma of maxillo - ethmoid region with intracranial extradural extention and its surgical management including repair of the skull base defect. MATERIAL : A 30 year female presented with progressive bilateral nasal obstruction, facial deformity for 5 years duration. She developed blindness in last 6 months. Recent CT s can showed large heterogeneous enhancing soft tissue mass in right maxillary sinus, nasal cavity and right ethmoid sinus invading the skull base . INTERVENTION : She underwent excision of the mass by modified weber ferguson incision and repair of skull base defect with temporalis muscle flap. Skin defect over the face and nose was repaired by median forehead flap. RESULT : There was total tumor clearance and no CSF leakage following surgery. CONCLUSION : Sinonasal malignancy with intracranial extradural extenti on is not a contraindication for successful surgical management. Resultant skull base defect can be repaired by a temporalis muscle flap to prevent CSF leak and intracranial infection

  19. UCE: A uracil excision (USERTM)-based toolbox for transformation of cereals

    DEFF Research Database (Denmark)

    Hebelstrup, Kim H; Christiansen, Michael W; Carciofi, Massimiliano;

    2010-01-01

    Background Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs...... (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient...

  20. Role of intraoperative 3D C-arm-based navigation in percutaneous excision of osteoid osteoma of long bones in children.

    Science.gov (United States)

    Rajasekaran, Shanmuganathan; Karthik, Karuppaiah; Chandra, Vattipalli Ravi; Rajkumar, Natesan; Dheenadhayalan, Jayaramaraju

    2010-03-01

    Failures of treatment of osteoid osteoma (OO) are related to errors in exact localization and incomplete excision of the nidus. We report the successful percutaneous excision of OO in five patients (upper end of femur - 3, tibia - 2). All patients had a minimally invasive reflective array fixed to the same bone followed by registration of anatomy by Iso-C three-dimensional (3D) C-arm. A tool navigator was used to plan the keyhole incision then a sleeve was introduced which allowed the usage of burr and curette to remove the tumor. After excision, the 3D C-arm was again used intraoperatively to confirm the complete eradication of the nidus. Adequate material for histology was obtained in four patients that confirmed the diagnosis of OO. In one child postexcision scans were successful in identifying incomplete removal requiring further excision of the nidus. All patients achieved excellent pain relief and were asymptomatic at an average follow-up of 3.2 years. 3D C-arm-based navigation offers the advantage of excellent localization, percutaneous excision, and intraoperative confirmation of adequate excision.

  1. Nucleotide excision repair in the test tube.

    NARCIS (Netherlands)

    N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

    1995-01-01

    textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

  2. Stoichiometry of base excision repair proteins correlates with increased somatic CAG instability in striatum over cerebellum in Huntington's disease transgenic mice.

    Science.gov (United States)

    Goula, Agathi-Vassiliki; Berquist, Brian R; Wilson, David M; Wheeler, Vanessa C; Trottier, Yvon; Merienne, Karine

    2009-12-01

    Huntington's disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5'-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5'-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLbeta was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLbeta strand displacement activity during LP-BER promotes the formation of stable 5'-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical

  3. Stoichiometry of base excision repair proteins correlates with increased somatic CAG instability in striatum over cerebellum in Huntington's disease transgenic mice.

    Directory of Open Access Journals (Sweden)

    Agathi-Vassiliki Goula

    2009-12-01

    Full Text Available Huntington's disease (HD is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5'-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1, the main enzyme responsible for 5'-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP-BER, were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLbeta was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLbeta strand displacement activity during LP-BER promotes the formation of stable 5'-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes

  4. Influence of XPB helicase on recruitment and redistribution of nucleotide excision repair proteins at sites of UV-induced DNA damage.

    Science.gov (United States)

    Oh, Kyu-Seon; Imoto, Kyoko; Boyle, Jennifer; Khan, Sikandar G; Kraemer, Kenneth H

    2007-09-01

    The XPB DNA helicase, a subunit of the basal transcription factor TFIIH, is also involved in nucleotide excision repair (NER). We examined recruitment of NER proteins in XP-B cells from patients with mild or severe xeroderma pigmentosum (XP) having different XPB mutations using local UV-irradiation through filters with 5 microm pores combined with fluorescent antibody labeling. XPC was rapidly recruited to UV damage sites containing DNA photoproducts (cyclobutane pyrimidine dimers, CPD) in all the XP-B and normal cells, thus reflecting its role in damage recognition prior to the function of XPB. Cells from the mild XP-B patients, with a missense mutation, showed delayed recruitment of all NER proteins except XPC to UV damage sites, demonstrating that this mutation impaired localization of these proteins. Surprisingly, in cells from severely affected patients, with a C-terminal XPB mutation, XPG and XPA proteins were normally recruited to UV damage sites demonstrating that this mutation permits recruitment of XPG and XPA. In marked contrast, in all the XP-B cells recruitment of XPF was absent immediately after UV and was delayed by 0.5 and 3 h in cells from the mild and severely affected XP patients, respectively. Redistribution of NER proteins was nearly complete in normal cells by 3 h but by 24 h redistribution was only partially present in cells from mild patients and virtually absent in cells from the severely affected patients. Ineffectual repair of UV-induced photoproducts resulting from delayed recruitment and impaired redistribution of NER proteins may contribute to the markedly increased frequency of skin cancer in XP patients.

  5. Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

    Science.gov (United States)

    Pan, Jishen; Sinclair, Elizabeth; Xuan, Zhuoli; Dyba, Marcin; Fu, Ying; Sen, Supti; Berry, Deborah; Creswell, Karen; Hu, Jiaxi; Roy, Rabindra; Chung, Fung-Lung

    2016-07-01

    The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125μM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA.

  6. Uracil Excision for Assembly of Complex Pathways

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Nielsen, Morten Thrane; Kim, Se Hyeuk

    2015-01-01

    Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most in...

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

    Science.gov (United States)

    Lee, Andrea J.; Wallace, Susan S.

    2016-11-01

    The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

  8. Loop electrosurgical excision of the cervix and subsequent risk for spontaneous preterm delivery: a population-based study of singleton deliveries during a 9-year period

    DEFF Research Database (Denmark)

    Noehr, Bugge; Jensen, Allan; Frederiksen, Kirsten;

    2009-01-01

    OBJECTIVE: Our aim was to assess the association between loop electrosurgical excision procedure (LEEP) and the subsequent risk for spontaneous preterm delivery, with the use of population-based data from various nationwide registries. STUDY DESIGN: The study population consisted of all singleton...

  9. The Mutyh base excision repair gene influences the inflammatory response in a mouse model of ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Ida Casorelli

    Full Text Available BACKGROUND: The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh(-/- mice to oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS. The Mutyh(-/- phenotype was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh(-/- mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh(-/- mice. Lymphoid hyperplasia and a significant reduction in Foxp3(+ regulatory T cells were observed only in Mutyh(-/- mice. CONCLUSIONS: The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.

  10. Submandibular approach for excision of a large schwannoma in the base of the tongue.

    Science.gov (United States)

    de Bree, R; Westerveld, G J; Smeele, L E

    2000-01-01

    A 24-year-old Turkish woman is described, who gradually developed progressive swallowing problems over 6 months due to a tumor in the base of the tongue. Magnetic resonance imaging showed a large well-circumscribed solid mass. Histopathological examination of an incisional biopsy showed a schwannoma. The tumor was completely removed through a submandibular approach. The postoperative course was uneventful and her complaints disappeared. The submandibular approach used gave an excellent exposure of the base of tongue with a less obvious scar than a lip-splitting incision.

  11. DNA Microarray-Based Diagnostics.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Evaluation of a cervical cancer screening program based on HPV testing and LLETZ excision in a low resource setting.

    Directory of Open Access Journals (Sweden)

    Margaret McAdam

    Full Text Available We conducted studies in Vanuatu to evaluate potential screening and treatment strategies to assist with control of cervical cancer. In a pilot study of 496 women, visual inspection and cytology were evaluated as screening tests for detection of CIN 2 or worse (CIN2+, observed in 21 of 206 subjects biopsied on the basis of abnormal visual inspection or cytology. Sensitivity of visual inspection with Lugol's Iodine for detection of CIN2+ on biopsy was 0.63, specificity was 0.32, and the positive predictive value was 0.09. For HSIL cytology, sensitivity was 0.99, specificity was 0.77, and the positive predictive value was 0.88. HSIL cytology was significantly more sensitive and had a significantly higher PPV for CIN 2+ than visual inspection (p<0.01. In a further study of 514 women, we compared testing for HR HPV and cytology as predictors of biopsy proven CIN 2+. Sensitivity of HSIL cytology for CIN2+ as established by loop excision of the cervix was 0.81, specificity was 0.94, and positive predictive value was 0.48. Sensitivity of a positive test for HR HPV for detection of CIN2+ was non-significantly different from cytology at 0.81, specificity was 0.94, and positive predictive value was 0.42. Combining the two tests gave a significantly lower sensitivity of 0.63, a specificity of 0.98, and a positive predictive value of 0.68. For women over 30 in a low resource setting without access to cytology, a single locally conducted test for high risk HPV with effective intervention could reduce cervical cancer risk as effectively as intervention based on cytology conducted in an accredited laboratory.

  14. "Blow-torch phenomenon" during laser assisted excision of a thyroglossal cyst at the base of the tongue

    Directory of Open Access Journals (Sweden)

    Anitha G Bhat

    2012-01-01

    Full Text Available We report a case of blow-torch phenomenon encountered during diode laser assisted excision of a thyroglossal cyst in a child. This is first such case report from India and highlights an unusual complication which anesthesiologists need to be aware of due to the increasing use of operative laser.

  15. Metallic Nanostructures Based on DNA Nanoshapes

    Directory of Open Access Journals (Sweden)

    Boxuan Shen

    2016-08-01

    Full Text Available 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.

  16. DNA Repair Gene Polymorphisms in Hereditary and Sporadic Breast Cancer

    Science.gov (United States)

    2006-03-01

    DNA polymerase beta, and DNA ligase 3. Alternatively, in long patch BER, few bases are excised and removed by FEN-1, including bases adjacent to...the damaged base, and incorporation of new nucleotides are mediated by PCNA, Polymerase delta or epsilon and DNA ligase I. 7 The nucleotide...requires the DNA-end-binding protein Ku, which binds free DNA ends and recruits DNA-PKcs. Xrcc4 is then recruited along with DNA ligase IV. The Rad50

  17. DNA-based applications in nanobiotechnology.

    Science.gov (United States)

    Abu-Salah, Khalid M; Ansari, Anees A; Alrokayan, Salman A

    2010-01-01

    Biological molecules such as deoxyribonucleic acid (DNA) have shown great potential in fabrication and construction of nanostructures and devices. The very properties that make DNA so effective as genetic material also make it a very suitable molecule for programmed self-assembly. The use of DNA to assemble metals or semiconducting particles has been extended to construct metallic nanowires and functionalized nanotubes. This paper highlights some important aspects of conjugating the unique physical properties of dots or wires with the remarkable recognition capabilities of DNA which could lead to miniaturizing biological electronics and optical devices, including biosensors and probes. Attempts to use DNA-based nanocarriers for gene delivery are discussed. In addition, the ecological advantages and risks of nanotechnology including DNA-based nanobiotechnology are evaluated.

  18. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    NARCIS (Netherlands)

    Biswas, Sukumar; Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Kok, Esther; Yuan, Zheng; Zhang, Dabing; Shi, Jianxin

    2016-01-01

    Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-tim

  19. Alternative DNA base pairing through metal coordination.

    Science.gov (United States)

    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.

  20. 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

    OpenAIRE

    Brown, Kyle L.; Roginskaya, Marina; Zou, Yue; Altamirano, Alvin; Basu, Ashis K.; Stone, Michael P.

    2009-01-01

    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 p...

  1. QPSO-Based Adaptive DNA Computing Algorithm

    Directory of Open Access Journals (Sweden)

    Mehmet Karakose

    2013-01-01

    Full Text Available 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.

  2. QPSO-based adaptive DNA computing algorithm.

    Science.gov (United States)

    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.

  3. DNA Coding Based Knowledge Discovery Algorithm

    Institute of Scientific and Technical Information of China (English)

    LI Ji-yun; GENG Zhao-feng; SHAO Shi-huang

    2002-01-01

    A novel DNA coding based knowledge discovery algorithm was proposed, an example which verified its validity was given. It is proved that this algorithm can discover new simplified rules from the original rule set efficiently.

  4. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available 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.

  5. DNA Sequence Optimization Based on Continuous Particle Swarm Optimization for Reliable DNA Computing and DNA Nanotechnology

    Directory of Open Access Journals (Sweden)

    N. K. Khalid

    2008-01-01

    Full Text Available Problem statement: In DNA based computation and DNA nanotechnology, the design of good DNA sequences has turned out to be an essential problem and one of the most practical and important research topics. Basically, the DNA sequence design problem is a multi-objective problem and it can be evaluated using four objective functions, namely, Hmeasure, similarity, continuity and hairpin. Approach: There are several ways to solve multi-objective problem, however, in order to evaluate the correctness of PSO algorithm in DNA sequence design, this problem is converted into single objective problem. Particle Swarm Optimization (PSO is proposed to minimize the objective in the problem, subjected to two constraints: melting temperature and GCcontent. A model is developed to present the DNA sequence design based on PSO computation. Results: Based on experiments and researches done, 20 particles are used in the implementation of the optimization process, where the average values and the standard deviation for 100 runs are shown along with comparison to other existing methods. Conclusion: The results achieve verified that PSO can suitably solves the DNA sequence design problem using the proposed method and model, comparatively better than other approaches.

  6. Luminescent DNA- and agar-based membranes.

    Science.gov (United States)

    Leones, R; Fernandes, M; Ferreira, R A S; Cesarino, I; Lima, J F; Carlos, L D; Bermudez, V de Zea; Magon, C J; Donoso, J P; Silva, M M; Pawlicka, A

    2014-09-01

    Luminescent materials containing europium ions are investigated for different optical applications. They can be obtained using bio-macromolecules, which are promising alternatives to synthetic polymers based on the decreasing oil resources. This paper describes studies of the DNA- and Agar-europium triflate luminescent membranes and its potential technological applications are expanded to electroluminescent devices. Polarized optical microscopy demonstrated that the samples are birefringent with submicrometer anisotropy. The X-ray diffraction analysis revealed predominantly amorphous nature of the samples and the atomic force microscopy images showed a roughness of the membranes of 409.0 and 136.1 nm for the samples of DNA10Eu and Agar1.11Eu, respectively. The electron paramagnetic resonance spectra of the DNA(n)Eu membranes with the principal lines at g ≈ 2.0 and g ≈ 4.8 confirmed uniform distribution of rare earth ions in a disordered matrix. Moreover, these strong and narrow resonance lines for the samples of DNA(n)Eu when compared to the Agar(n)Eu suggested a presence of paramagnetic radicals arising from the DNA matrix. The emission spectra suggested that the Eu3+ ions occupy a single local environment in both matrices and the excitation spectra monitored around the Eu emission lines pointed out that the Eu3+ ions in the Agar host were mainly excited via the broad band component rather than by direct intra-4f(6) excitation, whereas the opposite case occurred for the DNA-based sample.

  7. Excision of Selectable Markers Based on Inducible AlcR/alcA and Cre/loxP Systems%基于AlcR/alcA和Cre/loxP系统的标记基因诱导删除体系

    Institute of Scientific and Technical Information of China (English)

    赵青; 郭仰东; 谢华; 马荣才; 姚磊

    2011-01-01

    [Objective] To eliminate the potential risk in safety raised by selectable markers, an ethanol inducible excision system of selectable markers was constructed, which can be used during plant growth and development [ Method ] The selectable markers can be removed based on AlcR/alcA inducible system and Cre/loxP site-specific recombination system. In the presence of the exogenous inducer, the activation of the downstream Cre gene was enabled. Cre recombinase identified and catalyzed excision of the intervening sequence between two directly oriented loxP sites, including selectable marker gene, AlcR/alcA and Cre/loxP system. The gene of interest, gus, was constitutively expressed before and after induction. [ Result] The Arabidopsis thaliana transgenic plants were induced by ethanol. The results revealed that ethanol tightly control the "on" and "off' of the expression of Cre gene. After induction, the transgenic plants could not continuously grow on selective medium, which indicate the selectable marker was removed efficiently. The molecule analysis revealed the DNA fragment between two directly oriented loxP sites has been excised. [Conclusion] The results demonstrate that the excision of selectable markers based on inducible AlcR/alcA and Cre/loxP systems is reliable, and has a bright future.%[目的]通过构建能够在植物生长发育阶段经乙醇诱导将选择标记基因删除的载体,消除选择标记基因带来的潜在安全隐患.[方法]利用AlcR/alcA诱导系统和Cre/loxP位点特异性重组系统删除选择标记基因.当外源诱导物乙醇存在时,激活下游Cre的表达.Cre重组酶识别2个同向loxP位点,剔除位点之间的DNA片段,包括选择标记基因、AlcR/alcA系统和Cre/loxP系统,而目的基因gus在诱导前后均为组成型表达.[结果]拟南芥转基因植株受乙醇诱导严格控制Cre表达的“开”和“关”.经诱导的转基因植株不能在选择培养基上继续生长.

  8. Histone displacement during nucleotide excision repair

    DEFF Research Database (Denmark)

    Dinant, C.; Bartek, J.; Bekker-Jensen, S.

    2012-01-01

    Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER. © 2012 by the authors; licensee MDPI, Basel...

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

    Science.gov (United States)

    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

  10. Loop electrosurgical excision procedure for the treatment of cervical intraepithelial neoplasia: how much excision is enough?

    Science.gov (United States)

    Le, T; El-Sugi, R; Hicks-Boucher, W; Weberpals, J; Faught, W

    2013-08-01

    This is a retrospective observational study to compare outcomes in patients with cervical intraepithelial neoplasia (CIN) treated with loop electrosurgical excision procedure (LEEP) using combined ectocervical/endocervical resection vs ectocervical resection alone. We demonstrated that additional endocervical resection during loop electrosurgical excision procedure did not significantly lower the risk of subsequent recurrence compared with ectocervical resection alone, in the treatment of CIN. With current published data supporting subsequent increased adverse effects of LEEP on future obstetrical outcomes, endocervical excision should be applied selectively. We recommend that additional endocervical excision should be reserved only for patients with a strong suspicion of underlying endocervical canal involvement based on colposcopic assessment or in patients with unsatisfactory colposcopy, where it is essential to evaluate the endocervical canal.

  11. Role for DNA polymerase beta in response to ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Cramers, P.; Begg, A.C.; Vens, C.

    2007-01-01

    Evidence for a role of DNA polymerase beta in determining radiosensitivity is conflicting. In vitro assays show an involvement of DNA polymerase beta in single strand break repair and base excision repair of oxidative damages, both products of ionizing radiation. Nevertheless the lack of DNA polymer

  12. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  13. Communication: Electron ionization of DNA bases

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  15. DNA-Based Vaccine Protects Against Zika in Animal Study

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_161959.html DNA-Based Vaccine Protects Against Zika in Animal Study ... In animals infected with Zika virus, the synthetic DNA-based vaccine was 100 percent effective in protecting ...

  16. DNA-Based Vaccine Guards Against Zika in Monkey Study

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_161106.html DNA-Based Vaccine Guards Against Zika in Monkey Study ... THURSDAY, Sept. 22, 2016 (HealthDay News) -- An experimental DNA-based vaccine protected monkeys from infection with the ...

  17. Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Alexandra Vaisman

    2013-11-01

    Full Text Available Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER. We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8(th phosphodiester bond 5' and 4(th-5(th phosphodiester bonds 3' of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be

  18. [Uracil-DNA glycosylases].

    Science.gov (United States)

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  19. DNA & Protein detection based on microbead agglutination

    KAUST Repository

    Kodzius, Rimantas

    2012-06-06

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microparticles in the presence of a specific analyte thus enabling the macroscopic observation. Agglutination-based tests are most often used to explore the antibody-antigen reactions. Agglutination has been used for mode protein assays using a biotin/streptavidin two-component system, as well as a hybridization based two-component assay; however, as our work shows, two-component systems are prone to self-termination of the linking analyte and thus have a lower sensitivity. Three component systems have also been used with DNA hybridization, as in our work; however, their assay requires 48 hours for incubation, while our assay is performed in 5 minutes making it a real candidate for POC testing. We demonstrate three assays: a two-component biotin/streptavidin assay, a three-component hybridization assay using single stranded DNA (ssDNA) molecules and a stepped three-component hybridization assay. The comparison of these three assays shows our simple stepped three-component agglutination assay to be rapid at room temperature and more sensitive than the two-component version by an order of magnitude. An agglutination assay was also performed in a PDMS microfluidic chip where agglutinated beads were trapped by filter columns for easy observation. We developed a rapid (5 minute) room temperature assay, which is based on microbead agglutination. Our three-component assay solves the linker self-termination issue allowing an order of magnitude increase in sensitivity over two–component assays. Our stepped version of the three-component assay solves the issue with probe site saturation thus enabling a wider range of detection. Detection of the agglutinated beads with the naked eye by trapping in microfluidic channels has been shown.

  20. Nucleotide Excision Repair in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Hannes Lans

    2011-01-01

    Full Text Available Nucleotide excision repair (NER plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage.

  1. Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Akbari, Mansour; Sundheim, Ottar;

    2004-01-01

    Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (...

  2. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Science.gov (United States)

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  3. Structural basis of HIV-1 resistance to AZT by excision

    Energy Technology Data Exchange (ETDEWEB)

    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 (Rutgers); (Clark); (NCI)

    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.

  4. Strandwise translocation of a DNA glycosylase on undamaged DNA

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Yan; Nam, Kwangho; Spong, Marie C.; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L. (Harvard)

    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. Multidirectional Vector Excision Leads to Better Outcomes than Traditional Elliptical Excision of Facial Congenital Melanocytic Nevus

    Directory of Open Access Journals (Sweden)

    Seung Il Oh

    2013-09-01

    Full Text Available Background The elliptical excision is the standard method of removing benign skin lesions,such as congenital melanocytic nevi. This technique allows for primary closure, with little to nodog-ear deformity, but may sacrifice normal tissue adjacent to the lesion, resulting in scarswhich are unnecessarily long. This study was designed to compare the predicted results ofelliptical excision with those resulting from our excision technique.Methods Eighty-two patients with congenital melanocytic nevus on the face were prospectivelystudied. Each lesion was examined and an optimal ellipse was designed and marked onthe skin. After an incision on one side of the nevus margin, subcutaneous undermining wasperformed in the appropriate direction. The skin flap was pulled up and approximated alongseveral vectors to minimize the occurrence of dog-ear deformity.Results Overall, the final wound length was 21.1% shorter than that achieved by ellipticalexcision. Only 8.5% of the patients required dog-ear repair. There was no significant distortionof critical facial structures. All of the scars were deemed aesthetically acceptable based ontheir Patient and Observer Scar Assessment Scale scores.Conclusions When compared to elliptical excision, our technique appears to minimize dogeardeformity and decrease the final wound length. This technique should be considered analternative method for excision of facial nevi.

  6. DNA nanostructure-based imaging probes and drug carriers.

    Science.gov (United States)

    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.

  7. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao

    2011-01-01

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

  8. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2003-01-01

    Full Text Available DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT – the bionic wavelet transform (BWT – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the structural feature of the DNA sequence, was introduced into WT. It can adjust the weight value of each channel to maximise the useful energy distribution of the whole BWT output. The performance of the proposed BWT was examined by analysing synthetic and real DNA sequences. Results show that BWT performs better than traditional WT in presenting greater energy distribution. This new BWT method should be useful for the detection of the latent structural features in future DNA sequence analysis.

  9. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

    Directory of Open Access Journals (Sweden)

    Sozhamannan Shanmuga

    2006-04-01

    Full Text Available Abstract 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.

  10. Analytical Devices Based on Direct Synthesis of DNA on Paper.

    Science.gov (United States)

    Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

    2016-01-01

    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.

  11. [Retarded excision of pyrimidine dimers in human unstimulated lymphocytes].

    Science.gov (United States)

    Snopov, S A; Roza, L; de Gruijl, F R

    2006-01-01

    Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.

  12. New design of nucleotide excision repair (NER) inhibitors for combination cancer therapy.

    Science.gov (United States)

    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.

  13. Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guintini, Laetitia; Charton, Romain; Peyresaubes, François; Thoma, Fritz; Conconi, Antonio

    2015-12-01

    The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.

  14. A robust strategy for negative selection of Cre-loxP recombination-based excision of transgenes in induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Syandan Chakraborty

    Full Text Available Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs. For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In this study, we design an excisable loxP-flanked lentiviral construct that a includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1α promoter; b enables easy determination of lentiviral titer; c enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan negative selection; and d allows for transgene excision in a colony format. A reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.

  15. Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy.

    Science.gov (United States)

    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.

  16. How to make DNA count: DNA-based diagnostic tools in veterinary parasitology.

    Science.gov (United States)

    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.

  17. mtSSB may sequester UNG1 at mitochondrial ssDNA and delay uracil processing until the dsDNA conformation is restored

    DEFF Research Database (Denmark)

    Wollen Steen, Kristian; Doseth, Berit; westbye, Marianne;

    2012-01-01

    Single-strand DNA binding proteins protect DNA from nucleolytic damage, prevent formation of secondary structures and prevent premature reannealing of DNA in DNA metabolic transactions. In eukaryotes, the nuclear single-strand DNA binding protein RPA is essential for chromosomal DNA replication...... excision of uracil and oxidative demethylation of 3meC in single-stranded DNA by UNG1 and ABH1, respectively, whereas excision by NEIL1 was partially inhibited. mtSSB also effectively inhibited nicking of single-stranded DNA by APE1 and ABH1 and partially inhibited the lyase activity of NEIL1. Finally we...... identified a putative surface motif in mtSSB that may recruit UNG1 to DNA-bound mtSSB. We suggest that the massive amount of mtSSB in mitochondria effectively prevents processing of uracil and other types of damaged bases to avoid introduction of nicks in single-stranded mtDNA formed during replication...

  18. DNA Based Electrochromic and Photovoltaic Cells

    Science.gov (United States)

    2012-01-01

    and biodegradable material, has low cost and good film forming properties, is not toxic and forms transparent solutions with high viscosity [17]. An...applications requires fundamental studies on DNA in solid state, in which the behavior is expected to be different than that in solution. DNA is an acid , but...function of temperature of the blends samples of DNA with poly(ethylene dioxythiophene):poly(styrene sulphonate ) (PEDOT:PSS), poly(orthoethoxy aniline

  19. A novel bio-sensor based on DNA strand displacement.

    Directory of Open Access Journals (Sweden)

    Xiaolong Shi

    Full Text Available 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.

  20. DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

    Institute of Scientific and Technical Information of China (English)

    Luo Chunxiong; Mao Yongdong; Ouyang Qi

    2006-01-01

    Here we report a new method to detect DNA point mutations.The method is based on the formation and deformation of double-stranded DNA(dsDNA)membranes on a gold surface.It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface.In these systems,Fe(CN)63- was used as the reporter.As the temperature increases,a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears.At a special temperature,the and single base mutation target.Thus,the system provides a simple and sensitive method to detect DNA point mutations without labeling targets.

  1. Carbon-based electrode materials for DNA electroanalysis.

    Science.gov (United States)

    Kato, Dai; Niwa, Osamu

    2013-01-01

    This review addresses recent studies of newly developed carbon-based electrode materials and their use for DNA electroanalysis. Recently, new carbon materials including carbon nanotubes (CNT), graphene and diamond-based nanocarbon electrodes have been actively developed as sensing platforms for biomolecules, such as DNA and proteins. Electrochemical techniques using these new material-based electrodes can provide very simple and inexpensive sensing platforms, and so are expected to be used as one of the "post-light" DNA analysis methods, which include coulometric detection, amperometric detection with electroactive tags or intercalators, and potentiometric detection. DNA electroanalysis using these new carbon materials is summarized in view of recent advances on electrodes.

  2. Electroporation-based DNA delivery technology

    DEFF Research Database (Denmark)

    Gothelf, A; Gehl, Julie

    2014-01-01

    DNA delivery to for example skin and muscle can easily be performed with electroporation. The method is efficient, feasible, and inexpensive and the future possibilities are numerous. Here we present our protocol for gene transfection to mouse skin using naked plasmid DNA and electric pulses....

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

    Directory of Open Access Journals (Sweden)

    Kin Chan

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

  4. Hamilton Graph Based on DNA Computing

    Institute of Scientific and Technical Information of China (English)

    ZHANGJia-xiu

    2004-01-01

    DNA computing is a novel method for solving a class of intractable computationalproblems in which the computing can grow exponentially with problem size. Up to now, manyaccomplishments have been achieved to improve its performance and increase its reliability.Hamilton Graph Problem has been solved by means of molecular biology techniques. A smallgraph was encoded in molecules of DNA, and the “operations” of the computation wereperformed with standard protocols and enzymes. This work represents further evidence forthe ability of DNA computing to solve NP-complete search problems.

  5. Mitochondrial DNA diagnosis for taeniasis and cysticercosis.

    Science.gov (United States)

    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.

  6. Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group 2 mutants.

    NARCIS (Netherlands)

    M. van Duin (Mark); J.H. Janssen; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); L.H. Thompson; D. Bootsma (Dirk); A. Westerveld (Andries)

    1988-01-01

    textabstractThe human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In ord

  7. Controlling charge current through a DNA based molecular transistor

    Science.gov (United States)

    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.

  8. Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress

    DEFF Research Database (Denmark)

    Akbari, M; Otterlei, M; Pena Diaz, Javier;

    2007-01-01

    , these proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide......, indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence...... BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines....

  9. Antibody-controlled actuation of DNA-based molecular circuits

    Science.gov (United States)

    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.

  10. 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.

    Science.gov (United States)

    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

  11. 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

    Science.gov (United States)

    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.

  12. Lumbar disc excision through fenestration

    Directory of Open Access Journals (Sweden)

    Sangwan S

    2006-01-01

    Full Text Available Background : Lumbar disc herniation often causes sciatica. Many different techniques have been advocated with the aim of least possible damage to other structures while dealing with prolapsed disc surgically in the properly selected and indicated cases. Methods : Twenty six patients with clinical symptoms and signs of prolapsed lumbar intervertebral disc having radiological correlation by MRI study were subjected to disc excision by interlaminar fenestration method. Results : The assessment at follow-up showed excellent results in 17 patients, good in 6 patients, fair in 2 patients and poor in 1 patient. The mean preoperative and postoperative Visual Analogue Scores were 9.34 ±0.84 and 2.19 ±0.84 on scale of 0-10 respectively. These were statistically significant (p value< 0.001, paired t test. No significant complications were recorded. Conclusion : Procedures of interlaminar fenestration and open disc excision under direct vision offers sufficient adequate exposure for lumbar disc excision with a smaller incision, lesser morbidity, shorter convalescence, early return to work and comparable overall results in the centers where recent laser and endoscopy facilities are not available.

  13. The effect of base pair mismatch on DNA strand displacement

    CERN Document Server

    Broadwater, Bo

    2016-01-01

    DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single base pair mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data indicate that a single base pair mismatch in the invader stalls branch migration, and displacement occurs via direct dissociation of the destabilized incumbent strand from the substrate strand. We combined both branch migration and direct dissociation into a model, which we term, the concurrent displacement model, and used the first passage t...

  14. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    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

  15. DNA-based cryptographic methods for data hiding in DNA media.

    Science.gov (United States)

    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.

  16. Statistical mechanics of base stacking and pairing in DNA melting

    OpenAIRE

    Ivanov, Vassili; Zeng, Yan; Zocchi, Giovanni

    2004-01-01

    We propose a statistical mechanics model for DNA melting in which base stacking and pairing are explicitly introduced as distinct degrees of freedom. Unlike previous approaches, this model describes thermal denaturation of DNA secondary structure in the whole experimentally accessible temperature range. Base pairing is described through a zipper model, base stacking through an Ising model. We present experimental data on the unstacking transition, obtained exploiting the observation that at m...

  17. Unique magnetic signatures of mismatched base pairs in DNA

    Science.gov (United States)

    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.

  18. A novel DNA computing model based on RecA-mediated triple-stranded DNA structure

    Institute of Scientific and Technical Information of China (English)

    Fang Gang; Zhang Shemin; Dong Yafei; Xu Jin

    2007-01-01

    The field of DNA computing emerged in 1994 after Adleman's paper was published. Henceforth, a few scholars solved some noted NP-complete problems in this way. And all these methods of DNA computing are based on conventional Watson-Crick hydrogen bond of doublehelical DNA molecule. In this paper, we show that the triple-stranded DNA structure mediated by RecA protein can be used for solving computational problems. Sequence-specific recognition of double-stranded DNA by oligonucleotide-directed triple helix (triplex) formation is used to carry out the algorithm. We present procedure for the 3-vertex-colorability problems. In our proposed procedure, it is suggested that it is possible to solve more complicated problems with more variables by this model.

  19. DNA nanostructures based biosensor for the determination of aromatic compounds.

    Science.gov (United States)

    Gayathri, S Baby; Kamaraj, P; Arthanareeswari, M; Devikala, S

    2015-10-15

    Graphite electrode was modified using multi-walled carbon nanotubes (MWCNT), chitosan (CS), glutaraldehyde (GTA) and DNA nanostructures (nsDNA). DNA nanostructures of 50 nm in size were produced from single DNA template sequence using a simple two step procedure and were confirmed using TEM and AFM analysis. The modified electrode was applied to the electrochemical detection of aromatic compounds using EIS. The modified electrode was characterized using differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For comparison, electrochemical results derived from single stranded (50 bp length) and double stranded (50 bp length) DNA based biosensors were used. The results indicate that the modified electrode prior to nsDNA immobilization provides a viable platform that effectively promotes electron transfer between nsDNA and the electrode. The mode of binding between the nsDNA and aromatic compounds was investigated using EIS, indicating that the dominant interaction is non-covalent. nsDNA based biosensor was observed to act as an efficient biosensor in selective and sensitive identification of aromatic compounds.

  20. DNA-based tunable THz oscillator

    NARCIS (Netherlands)

    Malyshev, A. V.; Malyshev, V. A.; Dominguez-Adame, F.

    2009-01-01

    The intrinsic helix conformation of the DNA strands is known to be the key ingredient of control of the electric current through the molecule by the perpendicular (gate) electric field. We show theoretically that Bloch oscillations in periodic systems with helical conformation are also strongly affe

  1. Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis.

    Science.gov (United States)

    Bangun, A; Johnson, J L; Tripathy, D N

    1987-01-01

    DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-05

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

  3. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    Science.gov (United States)

    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.

  4. A liquid-crystal-based DNA biosensor for pathogen detection

    Science.gov (United States)

    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.

  5. A DNA based model for addition computation

    Institute of Scientific and Technical Information of China (English)

    GAO Lin; YANG Xiao; LIU Wenbin; XU Jin

    2004-01-01

    Much effort has been made to solve computing problems by using DNA-an organic simulating method, which in some cases is preferable to the current electronic computer. However, No one at present has proposed an effective and applicable method to solve addition problem with molecular algorithm due to the difficulty in solving the carry problem which can be easily solved by hardware of an electronic computer. In this article, we solved this problem by employing two kinds of DNA strings, one is called result and operation string while the other is named carrier. The result and operation string contains some carry information by its own and denotes the ultimate result while the carrier is just for carrying use. The significance of this algorithm is the original code, the fairly easy steps to follow and the feasibility under current molecular biological technology.

  6. Dynamic Simulation of Single DNA Molecule at the Base Level

    Institute of Scientific and Technical Information of China (English)

    LEI Xiao-Ling; WANG Xiao-Feng; HU Jun; FANG Hai-Ping

    2005-01-01

    @@ A mesoscopic discrete dsDNA model at the base level is proposed based on the statistical model (Phys. Rev. Lett.82 (1999) 4560). The numerical simulations reproduce the 65 pN plateau and those on the force vs extension for different supercoiling degrees are favourable with the experimental data. Our model has potential applications on the study of short DNA segments and provides a bridge between the statistical models and atomic modelling.

  7. How stable are the mutagenic tautomers of DNA bases?

    OpenAIRE

    Brovarets’ O. O.; Hovorun D. M.

    2010-01-01

    Aim. To determine the lifetime of the mutagenic tautomers of DNA base pairs through the investigation of the physicochemical mechanisms of their intramolecular proton transfer. Methods. Non-empirical quantum chemistry, the analysis of the electron density by means of Bader’s atom in molecules (AIM) theory and physicochemical kinetics were used. Results. Physicochemical character of the transition state of the intramolecular tautomerisation of DNA bases was investigated, the lifetime of mutage...

  8. Artifacts associated with the measurement of oxidized DNA bases.

    Science.gov (United States)

    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.

  9. A CLIQUE algorithm using DNA computing techniques based on closed-circle DNA sequences.

    Science.gov (United States)

    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.

  10. NMR analysis of base-pair opening kinetics in DNA

    Science.gov (United States)

    Szulik, Marta W.; Voehler, Markus; Stone, Michael P.

    2014-01-01

    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. PMID:25501592

  11. Cockayne Syndrome group B protein stimulates NEIL2 DNA glycosylase activity

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; Hvitby, Christina Poulsen; Popuri, Venkateswarlu

    2014-01-01

    excision repair and base excision repair. Here, we describe a new interaction partner for CSB, the DNA glycosylase NEIL2. Using both cell extracts and recombinant proteins, CSB and NEIL2 were found to physically interact independently of DNA. We further found that CSB is able to stimulate NEIL2 glycosylase...... in a DNA bubble structure using whole cell extracts. Taken together, our data supports a biological role for CSB and NEIL2 in transcription associated base excision repair.......Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide...

  12. Effect of radiotherapy on survival of women with locally excised ductal carcinoma in situ of the breast: a Surveillance, Epidemiology, and End Results population-based analysis

    Directory of Open Access Journals (Sweden)

    Qian GW

    2015-06-01

    Full Text Available Guo-Wei Qian,1,* Xiao-Jian Ni,1,* Zheng Wang,2 Yi-Zhou Jiang,1 Ke-Da Yu,1 Zhi-Ming Shao1 1Department of Breast Surgery, 2Department of Radiation Oncology, Shanghai Cancer Center and Cancer Institute, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Although it has been previously reported that radiotherapy (RT effectively reduced the incidence of local recurrence of ductal carcinoma in situ (DCIS following breast-conserving surgery (BCS, little is known about the effect of RT on survival of patients with locally excised DCIS. Patients and methods: Using Surveillance, Epidemiology, and End Results registry data, we selected 56,968 female DCIS patients treated with BCS between 1998 and 2007. Overall survival (OS and breast cancer-specific survival (BCSS were compared among patients who received RT or no RT using the Kaplan–Meier methods and Cox proportional hazards regression models. Results: Median follow-up was 91 months. In the multivariable model, patients receiving postoperative RT had better OS than those undergoing BCS alone (hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.53–0.67, P<0.001. This pattern remained after stratification by estrogen receptor (ER status and age. In contrast, RT delivery was not significantly associated with improved BCSS (HR 0.71, 95% CI 0.48–1.03, P=0.073. However, after stratifying by the above two variables, RT contributed to better BCSS in ER-negative/borderline patients (HR 0.41, 95% CI 0.19–0.88, P=0.023 and younger patients (≤50 years old; HR 0.37, 95% CI 0.15–0.91, P=0.030. Conclusion: Our analysis confirms the beneficial effect of RT on OS in women with locally excised DCIS and reveals the specific protective effect of RT on BCSS in ER-negative/borderline and younger patients. Keywords: ductal carcinoma in situ, breast cancer, breast-conserving surgery, radiotherapy, survival

  13. Charge Transport across DNA-Based Three-Way Junctions.

    Science.gov (United States)

    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.

  14. How stable are the mutagenic tautomers of DNA bases?

    Directory of Open Access Journals (Sweden)

    Brovarets’ O. O.

    2010-02-01

    Full Text Available Aim. To determine the lifetime of the mutagenic tautomers of DNA base pairs through the investigation of the physicochemical mechanisms of their intramolecular proton transfer. Methods. Non-empirical quantum chemistry, the analysis of the electron density by means of Bader’s atom in molecules (AIM theory and physicochemical kinetics were used. Results. Physicochemical character of the transition state of the intramolecular tautomerisation of DNA bases was investigated, the lifetime of mutagenic tautomers was calculated. Conclusions. The lifetime of the DNA bases mutagenic tautomers by 3–10 orders exceeds typical time of DNA replication in the cell (~103 s. This fact confirms that the postulate, on which the Watson-Crick tautomeric hypothesis of spontaneous transitions grounds, is adequate. The absence of intramolecular H-bonds in the canonical and mutagenic tautomeric forms determine their high stability

  15. Spectroscopic investigation on the telomeric DNA base sequence repeat

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Telomeres are protein-DNA complexes at the terminals of linear chromosomes, which protect chromosomal integrity and maintain cellular replicative capacity.From single-cell organisms to advanced animals and plants,structures and functions of telomeres are both very conservative. In cells of human and vertebral animals, telomeric DNA base sequences all are (TTAGGG)n. In the present work, we have obtained absorption and fluorescence spectra measured from seven synthesized oligonucleotides to simulate the telomeric DNA system and calculated their relative fluorescence quantum yields on which not only telomeric DNA characteristics are predicted but also possibly the shortened telomeric sequences during cell division are imrelative fluorescence quantum yield and remarkable excitation energy innerconversion, which tallies with the telomeric sequence of (TTAGGG)n. This result shows that telomeric DNA has a strong non-radiative or innerconvertible capability.``

  16. 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.

    Science.gov (United States)

    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.

  17. Endoscopic excision of cheek lipomas.

    Science.gov (United States)

    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.

    Science.gov (United States)

    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. PCR-based typing of DNA extracted from cigarette butts.

    Science.gov (United States)

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R

    1991-01-01

    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

  20. Direct DNA Analysis with Paper-Based Ion Concentration Polarization.

    Science.gov (United States)

    Gong, Max M; Nosrati, Reza; San Gabriel, Maria C; Zini, Armand; Sinton, David

    2015-11-01

    DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics.

  1. A novel image encryption algorithm based on DNA subsequence operation.

    Science.gov (United States)

    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.

  2. A Novel Image Encryption Algorithm Based on DNA Subsequence Operation

    Directory of Open Access Journals (Sweden)

    Qiang Zhang

    2012-01-01

    Full Text Available 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.

  3. A universal, photocleavable DNA base: nitropiperonyl 2'-deoxyriboside.

    Science.gov (United States)

    Pirrung, M C; Zhao, X; Harris, S V

    2001-03-23

    A universal, photochemically cleavable DNA base analogue would add desirable versatility to a number of methods in molecular biology. A novel C-nucleoside, nitropiperonyl deoxyriboside (NPdR, P), has been investigated for this purpose. NPdR can be converted to its 5'-DMTr-3'-CE-phosphoramidite and was incorporated into pentacosanucleotides by conventional synthesis techniques. The destabilizing effect on hybrid formation with a complementary strand when this P base opposes A, T, and G was found to be 3-5 kcal/mol, but 9 kcal/mol when it opposes C. Brief irradiation (lambda > 360 nm, 20 min) of DNA containing the P base and piperidine treatment causes strand cleavage giving the 3'- and 5'-phosphates. Two significant recent interests, universal/non-hydrogen-bonding base analogues and photochemical backbone cleavage, have thus been combined in a single molecule that serves as a light-based DNA scissors.

  4. A Rewritable, Random-Access DNA-Based Storage System

    Science.gov (United States)

    Tabatabaei Yazdi, S. M. Hossein; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-09-01

    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.

  5. ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2011-06-03

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

  7. Measurement of oxidatively generated base damage in cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-06-03

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

  8. Microwave-induced inactivation of DNA-based hybrid catalyst in asymmetric catalysis.

    Science.gov (United States)

    Zhao, Hua; Shen, Kai

    2016-03-01

    DNA-based hybrid catalysts have gained strong interests in asymmetric reactions. However, to maintain the high enantioselectivity, these reactions are usually conducted at relatively low temperatures (e.g. DNA-based hybrid catalyst even at low temperatures (such as 5 °C). Circular dichroism (CD) spectra and gel electrophoresis of DNA suggest that microwave exposure degrades DNA molecules and disrupts DNA double-stranded structures, causing changes of DNA-metal ligand binding properties and thus poor DNA catalytic performance.

  9. DNA nanotechnology based on i-motif structures.

    Science.gov (United States)

    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

  10. Base composition at mtDNA boundaries suggests a DNA triple helix model for human mitochondrial DNA large-scale rearrangements.

    Science.gov (United States)

    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.

  11. Application of DNA-based methods in forensic entomology.

    Science.gov (United States)

    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.

  12. Ultrasensitive electrochemical cocaine biosensor based on reversible DNA nanostructure.

    Science.gov (United States)

    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.

  13. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles.

    Science.gov (United States)

    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.

  14. Magnetophoretic-based microfluidic device for DNA Concentration.

    Science.gov (United States)

    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.

  15. Solid-State Nanopore-Based DNA Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Zewen Liu

    2016-01-01

    Full Text Available The solid-state nanopore-based DNA sequencing technology is becoming more and more attractive for its brand new future in gene detection field. The challenges that need to be addressed are diverse: the effective methods to detect base-specific signatures, the control of the nanopore’s size and surface properties, and the modulation of translocation velocity and behavior of the DNA molecules. Among these challenges, the realization of the high-quality nanopores with the help of modern micro/nanofabrication technologies is a crucial one. In this paper, typical technologies applied in the field of solid-state nanopore-based DNA sequencing have been reviewed.

  16. Ab initio Study of Naptho-Homologated DNA Bases

    Energy Technology Data Exchange (ETDEWEB)

    Sumpter, Bobby G [ORNL; Vazquez-Mayagoitia, Alvaro [ORNL; Huertas, Oscar [Universitat de Barcelona; Fuentes-Cabrera, Miguel A [ORNL; Orozco, Modesto [Institut de Recerca Biomedica, Parc Cientific de Barcelona, Barcelona, Spain; Luque, Javier [Universitat de Barcelona

    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. Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, Norbert [Department of Medicine, University of Alabama at Birmingham, Birmingham Alabama 35294; Zhukovskaya, Natalia [Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Bedwell, Gregory [Department of Microbiology, University of Alabama at Birmingham, Birmingham Alabama 35294; Nuth, Manunya [Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Gillilan, Richard [MacCHESS (Macromolecular Diffraction Facility at CHESS) Cornell University, Ithaca New York 14853; Prevelige, Peter E. [Department of Microbiology, University of Alabama at Birmingham, Birmingham Alabama 35294; Ricciardi, Robert P. [Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Abramson Cancer Center, School of Medicine, University of Pennsylvania, Philadelphia Pennsylvania 19104; Banerjee, Surajit [Department of Chemistry and Chemical Biology, Cornell University, and NE-CAT Argonne Illinois 60439; Chattopadhyay, Debasish [Department of Medicine, University of Alabama at Birmingham, Birmingham Alabama 35294

    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.

  18. Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

    Directory of Open Access Journals (Sweden)

    Md. Mahbubur Rahman

    2015-02-01

    Full Text Available 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.

  19. Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions

    NARCIS (Netherlands)

    C.E. Schrader; J. Vardo; E. Linehan; M.Z. Twarog; L.J. Niedernhofer (Laura); J. Stavnezer; J.H.J. Hoeijmakers (Jan)

    2004-01-01

    textabstractThe structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a

  20. Flow cytometry-based DNA hybridization and polymorphism analysis

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Poly(dimethylsiloxane) based microchip for DNA electrophoresis

    Institute of Scientific and Technical Information of China (English)

    LIU Changchun; CUI Dafu; WANG Li

    2004-01-01

    A novel poly(dimethylsiloxane)(PDMS) -based microchip for DNA separation through electrophoresis has been developed using a micro-electro-mechanical-system(MEMS) technology. Unlike previous hybrid PDMS microchip, one PDMS film is first created on glass support by pressing method in our microchip. Thus, increased band-broadening phenomena, arising from the material nonuniformity at the walls of microchannel, can be avoided in electrophoresis process. A low-viscosity hydroxypropylmethylcellulose-100 (HPMC-100) is used as the separation medium for fluorescent intercalator-labeled double-stranded DNA (dsDNA) fragments. Mannitol is introduced to PDMS-based microchip as a separation medium additive to enhance separation efficiency. At applied electric field strength of 150 V/cm, excellent separations of the PCR marker could be achieved with an effective separation distance of 25mm .

  2. Statistical mechanics of base stacking and pairing in DNA melting.

    Science.gov (United States)

    Ivanov, Vassili; Zeng, Yan; Zocchi, Giovanni

    2004-11-01

    We propose a statistical mechanics model for DNA melting in which base stacking and pairing are explicitly introduced as distinct degrees of freedom. Unlike previous approaches, this model describes thermal denaturation of DNA secondary structure in the whole experimentally accessible temperature range. Base pairing is described through a zipper model, base stacking through an Ising model. We present experimental data on the unstacking transition, obtained exploiting the observation that at moderately low pH this transition is moved down to experimentally accessible temperatures. These measurements confirm that the Ising model approach is indeed a good description of base stacking. On the other hand, comparison with the experiments points to the limitations of the simple zipper model description of base pairing.

  3. The recombinase IntA is required for excision of esp-containing ICEEfm1 in Enterococcus faecium.

    Science.gov (United States)

    Top, Janetta; Sinnige, Jan C; Majoor, Eline A M; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

    2011-02-01

    Comparative genome analysis of Enterococcus faecium recently revealed that a genomic island containing the esp gene, referred to as the esp-containing pathogenicity island (esp PAI), can be transferred by conjugation and contains a partial Tn916-like element and an integrase gene, intA. Here, we characterize the role of intA in the excision of the esp PAI. An intA insertion-deletion mutant in E. faecium E1162 (E1162ΔintA) was constructed and in trans complemented with wild-type intA (E1162ΔintA::pEF30). Circular intermediates (CI) of excised esp PAI were determined using inverse PCR analysis on purified chromosomal DNA from strains E1162, E1162Δesp, E1162ΔintA, and E1162ΔintA::pEF30. In E1162 and E1162Δesp, CI of the esp PAI were detected. No CI were detected in E1162ΔintA, while in the complemented strain E1162ΔintA::pEF30 CI formation was restored, indicating that intA is essential for excision and subsequent mobilization of the esp-containing genomic island in E. faecium. Based on the fact that this island can be mobilized and is self-transmissible, we propose to change the name of the esp PAI to ICEEfm1.

  4. DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites.

    Science.gov (United States)

    López-Camarillo, César; Lopez-Casamichana, Mavil; Weber, Christian; Guillen, Nancy; Orozco, Esther; Marchat, Laurence A

    2009-12-01

    Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.

  5. DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories.

    Science.gov (United States)

    Vago, Riccardo; Leva, Valentina; Biamonti, Giuseppe; Montecucco, Alessandra

    2009-08-15

    DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.

  6. DNA methylation detection based on difference of base content

    Science.gov (United States)

    Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

    2016-04-01

    Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

  7. Impact of cigarette taxation policy on excise revenues and cigarette consumption in Uzbekistan

    Directory of Open Access Journals (Sweden)

    Konstantin S. Krasovsky

    2013-05-01

    Full Text Available BACKGROUND: In 2012, Uzbekistan ratified the Framework Convention on Tobacco Control, which states that price and tax measures are an effective means of reducing tobacco consumption. We aimed to explore the effect of taxation policies on revenues and cigarette consumption. METHODS: Data on tax rates, revenues, cigarette sales were taken from national reports. To forecast potential revenues, a scenario analysis was performed. RESULTS: In 1991-2004, ad valorem excise system was in place in Uzbekistan, which was later replaced by the specific excise system. In 1997-2011, the nominal average excise has increased by a factor of twenty, but in real terms, after a sharp increase in 1999, average excise declined annually and increased only in 2010-2011. Annual cigarette sales per capita of adult population in 1999-2007 constituted 17-25 cigarette packs, while in 2008-2011 it increased to 30-37 packs. Four scenarios of excise tax increases in 2012 were developed: one actual scenario based on the rates effective in Uzbekistan in 2012, and three hypothetical ones anticipating excise rates increase by 1.5, 2 and 3-fold. With actual excise increase in 2012, the inflation-adjusted budget revenues would grow by 5%, and with three hypothetical - by 17%, 35% and 66% respectively, despite the decline of tax-paid cigarette sales. CONCLUSION: Stabilization or reduction in cigarette excises in Uzbekistan in 2002-2008 led to a decline in real excise revenues and the growth of cigarette sales. In 1999 and 2010-2011, excises were significantly increased and the real revenues have risen, despite the decline in cigarette sales. As cigarette prices are low, the illegal outflow of cigarettes from Uzbekistan apparently exceeds the illegal inflow. A significant increase in cigarette excise (1.5-3 fold can both increase budget revenues and reduce cigarette consumption, with greater increase yielding more benefits.

  8. Indicator Based and Indicator - Free Electrochemical DNA Biosensors

    Science.gov (United States)

    2007-11-02

    of genomic material from infectious organisms. Methylene blue (MB) is an aromatic heterocycle that binds strongly to DNA via intercalation. MB...detection of disease- related point mutation in the guanine bases of the cyanobacteria . The resulting biosensors offer great promise for mismatch

  9. Mitochondrial and chloroplast DNA based phylogeny of Pelargonium (Geraniaceae)

    NARCIS (Netherlands)

    Bakker, F.T.; Culham, A.; Pankhurst, C.E.; Gibby, M.

    2000-01-01

    Overall phylogenetic relationships within the genus Pelargonium (Geraniaceae) were inferred based on DNA sequences from mitochondrial(mt)-encoded nad1 b/c exons and from chloroplast(cp)-encoded trnL (UAA) 5' exon-trnF (GAA) exon regions using two species of Geranium and Sarcocaulon vanderetiae as ou

  10. Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Yun Fei BAI; Qin Yu GE; Tong Xiang LI; Jin Ke WANG; Quan Jun LIU; Zu Hong LU

    2005-01-01

    The double-stranded DNA (dsDNA) probe contains two different protein binding sites.One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme.The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

  11. Karyotyping of Brassica oleracea L.based on rDNA and Cot-1 DNA fluorescence in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    WANG Taixia; WU Chunhong; HUANG Jinyong; WEI Wenhui

    2007-01-01

    To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.

  12. Base excision repair efficiency and mechanism in nuclear extracts are influenced by the ratio between volume of nuclear extraction buffer and nuclei-Implications for comparative studies

    DEFF Research Database (Denmark)

    Akbari, Mansour; Krokan, Hans E

    2012-01-01

    using purified proteins essentially mirror properties of the proteins used, and does not necessarily reflect the mechanism as it occurs in the cell. Nuclear extracts from cultured cells have the capacity to carry out complete BER and can give important information on the mechanism. Furthermore......, candidate proteins in extracts can be inhibited or depleted in a controlled way, making defined extracts an important source for mechanistic studies. The major drawback is that there is no standardized method of preparing nuclear extract for BER studies, and it does not appear to be a topic given much...... attention. Here we have examined BER activity of nuclear cell extracts from HeLa cells, using as substrate a circular DNA molecule with either uracil or an AP-site in a defined position. We show that BER activity of nuclear extracts from the same batch of cells varies inversely with the volume of nuclear...

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

    Science.gov (United States)

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

    2015-08-06

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

  14. Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set.

    Science.gov (United States)

    Krueger, Andrew T; Kool, Eric T

    2008-03-26

    We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis

  15. DNA-Based Self-Assembly of Fluorescent Nanodiamonds.

    Science.gov (United States)

    Zhang, Tao; Neumann, Andre; Lindlau, Jessica; Wu, Yuzhou; Pramanik, Goutam; Naydenov, Boris; Jelezko, Fedor; Schüder, Florian; Huber, Sebastian; Huber, Marinus; Stehr, Florian; Högele, Alexander; Weil, Tanja; Liedl, Tim

    2015-08-12

    As a step toward deterministic and scalable assembly of ordered spin arrays we here demonstrate a bottom-up approach to position fluorescent nanodiamonds (NDs) with nanometer precision on DNA origami structures. We have realized a reliable and broadly applicable surface modification strategy that results in DNA-functionalized and perfectly dispersed NDs that were then self-assembled in predefined geometries. With optical studies we show that the fluorescence properties of the nitrogen-vacancy color centers in NDs are preserved during surface modification and DNA assembly. As this method allows the nanoscale arrangement of fluorescent NDs together with other optically active components in complex geometries, applications based on self-assembled spin lattices or plasmon-enhanced spin sensors as well as improved fluorescent labeling for bioimaging could be envisioned.

  16. Bisulfite-based epityping on pooled genomic DNA provides an accurate estimate of average group DNA methylation

    Directory of Open Access Journals (Sweden)

    Docherty Sophia J

    2009-03-01

    Full Text Available Abstract Background 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 relatively large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. We propose that a high-throughput DNA pooling approach will facilitate the use of emerging methylomic profiling techniques in large samples. Results Compared with data generated from 89 individual samples, our analysis of 205 CpG sites spanning nine independent regions of the genome demonstrates that DNA pools can be used to provide an accurate and reliable quantitative estimate of average group DNA methylation. Comparison of data generated from the pooled DNA samples with results averaged across the individual samples comprising each pool revealed highly significant correlations for individual CpG sites across all nine regions, with an average overall correlation across all regions and pools of 0.95 (95% bootstrapped confidence intervals: 0.94 to 0.96. Conclusion In this study we demonstrate the validity of using pooled DNA samples to accurately assess group DNA methylation averages. Such an approach can be readily applied to the assessment of disease phenotypes reducing the time, cost and amount of DNA starting material required for large-scale epigenetic analyses.

  17. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    Science.gov (United States)

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  18. Nucleotide excision repair in differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

    2007-01-03

    Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

  19. Rapid sequencing of DNA based on single-molecule detection

    Science.gov (United States)

    Soper, Steven A.; Davis, Lloyd M.; Fairfield, Frederick R.; Hammond, Mark L.; Harger, Carol A.; Jett, James H.; Keller, Richard A.; Marrone, Babetta L.; Martin, John C.; Nutter, Harvey L.; Shera, E. Brooks; Simpson, Daniel J.

    1991-07-01

    Sequencing the human genome is a major undertaking considering the large number of nucleotides present in the genome and the slow methods currently available to perform the task. The authors have recently reported on a scheme to sequence DNA rapidly using a non-gel based technique. The concept is based upon the incorporation of fluorescently labeled nucleotides into a strand of DNA, isolation and manipulation of a labeled DNA fragment and the detection of single nucleotides using ultra-sensitive laser-induced fluorescence detection following their cleavage from the fragment. Detection of individual fluorophores in the liquid phase was accomplished with time-gated detection following pulsed-laser excitation. The photon bursts from individual rhodamine 6G (R6G) molecules travelling through a laser beam have been observed, as have bursts from single fluorescently modified nucleotides. Using two different biotinylated nucleotides as a model system for fluorescently labeled nucleotides, the authors have observed synthesis of the complementary copy of M13 bacteriophage. Work with fluorescently labeled nucleotides is underway. Individual molecules of DNA attached to a microbead have been observed and manipulated with an epifluorescence microscope.

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

    Science.gov (United States)

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

    2013-09-17

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

  1. Three types of abdominoperineal excision procedures for the rectal cancer based on anatomic landmarks classification%基于解剖边界划分的三种直肠癌腹会阴联合切除术式

    Institute of Scientific and Technical Information of China (English)

    叶颖江; 申占龙; 王杉

    2014-01-01

    对于肿瘤位置过低、肿瘤明显外侵和骨盆过于狭小的患者,腹会阴联合切除术(APE)依然是主要术式。APE腹部操作已明确应遵循全直肠系膜切除术( TME )原则,但会阴操作原则尚未达成共识,其重要原因在于会阴部操作缺乏明确的解剖边界,以至于难以实现标准化。2014年,瑞典外科学家Holm教授基于会阴区筋膜、神经和血管组成的解剖边界,提出了直肠癌APE的术式分类新概念,将APE分为3类,即括约肌间APE、肛提肌外APE和坐骨肛管间APE。此新概念的提出,使APE术式分类更明确,解剖界标更清晰,更利于推广和标准化。本文结合文献和笔者的诊治经验,对这3种术式分别加以介绍和讨论。%Abdominoperineal excision (APE) procedure is still the main approach to low rectal cancer patients with short distance from the anal verge, obvious invasion of adjacent organs and narrow pelvis. Although the principle of TME (total mesorectal excision) needs to be obeyed in the abdominal phase of APE procedure, it does not reach the consensus for the perineal phase. The important reason is the lack of definite anatomic landmarks in the perineal phase, thus the standardization of the procedure remains hard. In 2014, Swedish surgeon, professor Holm, proposed the new conception to classify the APE procedure into three types, which were intersphincteric APE, the extralavator APE and the ischioanal APE, based on the anatomic landmarks with perineal fascias, nervous and blood vessels. In this paper, we combine the review of literatures and our experiences of treatment to introduce and discuss these three types of APE procedures. This new concep is based on anatomic landmarks which makes the category of APE procedure more definitive, the anatomic dissection more clear and the standardization and adoption of APE procedure much easier.

  2. Giant rhinophyma: Excision with coblation assisted surgery.

    Science.gov (United States)

    Sahin, Caner; Turker, Mesut; Celasun, Bulent

    2014-01-01

    An 83-year-old man presented with an unusually severe case of rhinophyma. Giant rhinopyhma is very rare in literature. The giant lesion was widely excised using sharp surgical incision and coblation assisted surgery. Using direct coblation to the nasal dorsum may cause edema in the surrounding tissue. There was minimal edema in surrounding tissue using this technique. A full thickness-skin graft was applied after excision. Cosmetic and functional postoperative results were satisfactory.

  3. Giant rhinophyma: Excision with coblation assisted surgery

    Directory of Open Access Journals (Sweden)

    Caner Sahin

    2014-01-01

    Full Text Available An 83-year-old man presented with an unusually severe case of rhinophyma. Giant rhinopyhma is very rare in literature. The giant lesion was widely excised using sharp surgical incision and coblation assisted surgery. Using direct coblation to the nasal dorsum may cause edema in the surrounding tissue. There was minimal edema in surrounding tissue using this technique. A full thickness-skin graft was applied after excision. Cosmetic and functional postoperative results were satisfactory.

  4. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology

    Institute of Scientific and Technical Information of China (English)

    Sarah C Shuck; Emily A Short; John J Turchi

    2008-01-01

    Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

  5. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase.

    Science.gov (United States)

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu

    2010-02-01

    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  6. Detection Tuna and Processed Products Based Protein and DNA Barcoding

    Directory of Open Access Journals (Sweden)

    Nuring Wulansari

    2015-11-01

    Full Text Available Tuna is the second largest fishery commodity in Indonesia after the shrimp. Since the high demand and the limited stock of tuna resulted in fraudulent chance. Authentication is required to meassure consumers regarding the accuracy of its labeling and food safety. In this study, the authentication was based on protein and DNA barcoding using cytochrome-b gene (cyt-b of the mitochondrial DNA as the target of gene. Primer of cyt b gene was designed based on the tuna species. This study aimed to identify the authenticity of tuna fresh and its processed products through protein using SDS-PAGE and DNA barcoding techniques. The phases of this research were protein electrophoresis by SDS-PAGE, DNA extraction, PCR amplification, electrophoresis and sequencing. Samples of fresh fish (Tu1, Tu2, Tu3, Tu4, and Tu5 and processed tuna (canned and steak were successfully extracted. Result showed that SDS-PAGE proved the damage of proteins in the processed tuna, so this method was not appropriate if it is used to identify the authenticity of tuna. PCR electrophoresis results showed that the samples of tuna, tuna steak, sushi, meat ball, abon, and caned tuna were successfully amplified in the range of 500-750 bp except Ka3, which was in line with the target of DNA (620 bp. Resulted sequences of Tu2, Tu3, Tu4 and Tu5 were identified according the results of morphometric namely T. albacares, while Tu1 was identified as T. obesus with homology level of 99%. Processed tunas (steak and canned tuna were identified as T. albacares, as stated on the labels.

  7. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

    2010-08-01

    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  8. A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

    Science.gov (United States)

    De, Ananya; Campbell, Colin

    2007-02-15

    The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

  9. Progress of DNA-based Methods for Species Identification

    Institute of Scientific and Technical Information of China (English)

    HU Zhen; ZHANG Su-hua; WANG Zheng; BIAN Ying-nan; LI Cheng-tao

    2015-01-01

    Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The cur-rent reviewshows common target genes and screening criteria suitable for species identification, and de-scribed various DNA-based molecular biology methods about species identification. Additionally, it dis-cusses the future development of species identification combined with real-time PCR and sequencing technologies.

  10. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  11. Direct Electrical Detection of DNA Hybridization Based on Electrolyte-Gated Graphene Field-Effect Transistor

    Science.gov (United States)

    Ohno, Yasuhide; Okamoto, Shogo; Maehashi, Kenzo; Matsumoto, Kazuhiko

    2013-11-01

    DNA hybridization was electrically detected by graphene field-effect transistors. Probe DNA was modified on the graphene channel by a pyrene-based linker material. The transfer characteristic was shifted by the negative charges on the probe DNA, and the drain current was changed by the full-complementary DNA while no current change was observed after adding noncomplementary DNA, indicating that the graphene field-effect transistor detected the DNA hybridization. In addition, the number of DNAs was estimated by the simple plate capacitor model. As a result, one probe DNA was attached on the graphene channel per 10×10 nm2, indicating their high density functionalization. We estimated that 30% of probe DNA on the graphene channel was hybridized with 200 nM full-complementary DNA while only 5% of probe DNA was bound to the noncomplementary DNA. These results will help to pave the way for future biosensing applications based on graphene FETs.

  12. Nucleotide excision repair in intact cells contrasts with high dual incision activity in vitro

    NARCIS (Netherlands)

    Jansen, J.; Olsen, A.K.; Wiger, R.; Naegeli, H.; Boer, de P.; Hoeven, van der F.; Holme, J.A.; Brunborg, G.; Mullenders, L.

    2001-01-01

    The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, gl

  13. Measurement of oxidative DNA damage by gas chromatography-mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases.

    Science.gov (United States)

    Jenner, A; England, T G; Aruoma, O I; Halliwell, B

    1998-04-15

    Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause 'artifactual' oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 degrees C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC-MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 degrees C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54-59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatization conditions used. Only hydrolysed DNA samples that were derivatized at 23 degrees C in the presence of ethanethiol produced 8-hydroxyguanine levels (56+/-8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 degrees C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC-MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 degrees C, removing air from the

  14. Mitochondrial DNA maintenance: an appraisal.

    Science.gov (United States)

    Akhmedov, Alexander T; Marín-García, José

    2015-11-01

    Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS), and Ca(2+) handling to stress responses, cell survival, and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular disorders, cancer, premature aging, and cardiovascular diseases, including myocardial ischemia, cardiomyopathy, and heart failure. Mitochondria are unique as they contain their own genome organized into DNA-protein complexes, so-called mitochondrial nucleoids, along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription, and repair. Although the organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair, and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to oxidative stress and other types of mtDNA damage. Defects in the components of these highly organized machineries, which mediate mtDNA maintenance (replication and repair), may result in accumulation of point mutations and/or deletions in mtDNA and decreased mtDNA copy number impairing mitochondrial function. This review will focus on the mechanisms of mtDNA maintenance with emphasis on the proteins implicated in these processes and their functional role in various disease conditions and aging.

  15. A Graphene-Based Biosensing Platform Based on Regulated Release of an Aptameric DNA Biosensor.

    Science.gov (United States)

    Mao, Yu; Chen, Yongli; Li, Song; Lin, Shuo; Jiang, Yuyang

    2015-11-09

    A novel biosensing platform was developed by integrating an aptamer-based DNA biosensor with graphene oxide (GO) for rapid and facile detection of adenosine triphosphate (ATP, as a model target). The DNA biosensor, which is locked by GO, is designed to contain two sensing modules that include recognition site for ATP and self-replication track that yields the nicking domain for Nt.BbvCI. By taking advantage of the different binding affinity of single-stranded DNA, double-stranded DNA and aptamer-target complex toward GO, the DNA biosensor could be efficiently released from GO in the presence of target with the help of a complementary DNA strand (CPDNA) that partially hybridizes to the DNA biosensor. Then, the polymerization/nicking enzyme synergetic isothermal amplification could be triggered, leading to the synthesis of massive DNA amplicons, thus achieving an enhanced sensitivity with a wide linear dynamic response range of four orders of magnitude and good selectivity. This biosensing strategy expands the applications of GO-DNA nanobiointerfaces in biological sensing, showing great potential in fundamental research and biomedical diagnosis.

  16. Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct.

    Science.gov (United States)

    Politica, Dustin A; Malik, Chanchal K; Basu, Ashis K; Stone, Michael P

    2015-12-21

    3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-d

  17. Main features of DNA-based immunization vectors

    Directory of Open Access Journals (Sweden)

    V. Azevedo

    1999-02-01

    Full Text Available DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them, polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

  18. 碱基切除修复基因HOGG1特异性锤头状核酶表达载体的构建及其功能的初步研究%Constructing the Eukaryotic Expression Vector to Study Preliminarily the Functions of Hammerhead Ribozyme Targeting Base Excision Repair Gene HOGG1

    Institute of Scientific and Technical Information of China (English)

    张遵真; 张勤; 吴媚

    2006-01-01

    Objective Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 cells would be investigated. Methods According to computer design, two specific restriction site BamHⅠ and EcoRⅠ were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by ampicillin resistance, and plasmids were extracted from the positive recombinants and digested by BamH Ⅰ and EcoR Ⅰ, and then were analyzed by agarose gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. Results The recombinants containing the ribozyme gene

  19. DNA-based influenza vaccines as immunoprophylactic agents toward universality.

    Science.gov (United States)

    Zhang, Han; El Zowalaty, Mohamed E

    2016-01-01

    Influenza is an illness of global public health concern. Influenza viruses have been responsible for several pandemics affecting humans. Current influenza vaccines have proved satisfactory safety; however, they have limitations and do not provide protection against unexpected emerging influenza virus strains. Therefore, there is an urgent need for alternative approaches to conventional influenza vaccines. The development of universal influenza vaccines will help alleviate the severity of influenza pandemics. Influenza DNA vaccines have been the subject of many studies over the past decades due to their ability to induce broad-based protective immune responses in various animal models. The present review highlights the recent advances in influenza DNA vaccine research and its potential as an affordable universal influenza vaccine.

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

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning......DNA-damage response and repair are crucial to maintain genetic stability, and are consequently considered central to aging and longevity. Here, we investigate whether this pathway overall associates to longevity, and whether specific sub-processes are more strongly associated with longevity than...... and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10-5), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using...

  1. Control of Staphylococcus aureus pathogenicity island excision.

    Science.gov (United States)

    Mir-Sanchis, Ignacio; Martínez-Rubio, Roser; Martí, Miguel; Chen, John; Lasa, Íñigo; Novick, Richard P; Tormo-Más, María Ángeles; Penadés, José R

    2012-09-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15-17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small-headed phage-like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI-encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI-coded Xis is absolutely required for recombination between the SaPI att(L) and att(R) sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.

  2. Proximal clavicle excision: an analysis of results.

    Science.gov (United States)

    Acus, R W; Bell, R H; Fisher, D L

    1995-01-01

    Medial clavicle excision has been reported by several authors, but few cases are documented, and long-term follow-up information is lacking. The purpose of this study was to examine the long-term results of medial clavicle excision in regard to function, pain, cosmesis, and complications. Fifteen patients ranging in age from 18 to 64 years (average 43 years) were evaluated an average of 4.6 years (range 1 to 14 years) after proximal clavicle excision. The indications for excision were unstable anterior subluxation/dislocation of the sternoclavicular joint (four cases), unstable posterior dislocation (one case), sternoclavicular osteoarthritis (nine cases), and proximal clavicle osteomyelitis (one case). An average of 2.9 cm of the medial clavicle was excised (range 1 to 4 cm). Fourteen of the 15 patients received significant relief of pain. On a strict grading scale four patients had an excellent result, five a good result, four a fair result, and two a poor result. Regeneration of the clavicle appeared to contribute to a poor result. No operative complications occurred. These findings aid our understanding of surgical options and outcome in the treatment of sternoclavicular joint disease.

  3. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    Science.gov (United States)

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.

  4. Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells.

    Science.gov (United States)

    Lefevre, Sophie; Brossas, Caroline; Auchère, Françoise; Boggetto, Nicole; Camadro, Jean-Michel; Santos, Renata

    2012-09-15

    Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H(2)O(2). On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.

  5. Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation.

    Science.gov (United States)

    Sampoli Benítez, Benedetta A; Arora, Karunesh; Balistreri, Lisa; Schlick, Tamar

    2008-12-31

    DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X

  6. Physics of base-pairing dynamics in DNA

    Science.gov (United States)

    Manghi, Manoel; Destainville, Nicolas

    2016-05-01

    As a key molecule of life, Deoxyribo-Nucleic Acid (DNA) is the focus of numbers of investigations with the help of biological, chemical and physical techniques. From a physical point of view, both experimental and theoretical works have brought quantitative insights into DNA base-pairing dynamics that we review in this Report, putting emphasis on theoretical developments. We discuss the dynamics at the base-pair scale and its pivotal coupling with the polymer one, with a polymerization index running from a few nucleotides to tens of kilo-bases. This includes opening and closure of short hairpins and oligomers as well as zipping and unwinding of long macromolecules. We review how different physical mechanisms are either used by Nature or utilized in biotechnological processes to separate the two intertwined DNA strands, by insisting on quantitative results. They go from thermally-assisted denaturation bubble nucleation to force- or torque-driven mechanisms. We show that the helical character of the molecule, possibly supercoiled, can play a key role in many denaturation and renaturation processes. We categorize the mechanisms according to the relative timescales associated with base-pairing and chain orientational degrees of freedom such as bending and torsional elastic ones. In some specific situations, these chain orientational degrees of freedom can be integrated out, and the quasi-static approximation is valid. The complex dynamics then reduces to the diffusion in a low-dimensional free-energy landscape. In contrast, some important cases of experimental interest necessarily appeal to far-from-equilibrium statistical mechanics and hydrodynamics.

  7. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair.

    Science.gov (United States)

    Chen, Xi; Zhong, Shijun; Zhu, Xiao; Dziegielewska, Barbara; Ellenberger, Tom; Wilson, Gerald M; MacKerell, Alexander D; Tomkinson, Alan E

    2008-05-01

    Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

  8. Changes in DNA repair during aging

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei; Mao, Zhiyong; Hine, Christpher

    2007-01-01

    DNA is a precious molecule. It encodes vital information about cellular content and function. There are only two copies of each chromosome in the cell, and once the sequence is lost no replacement is possible. The irreplaceable nature of the DNA sets it apart from other cellular molecules, and makes it a critical target for age-related deterioration. To prevent DNA damage cells have evolved elaborate DNA repair machinery. Paradoxically, DNA repair can itself be subject to age-related changes and deterioration. In this review we will discuss the changes in efficiency of mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER) and double-strand break (DSB) repair systems during aging, and potential changes in DSB repair pathway usage that occur with age. Mutations in DNA repair genes and premature aging phenotypes they cause have been reviewed extensively elsewhere, therefore the focus of this review is on the comparison of DNA repair mechanisms in young versus old. PMID:17913742

  9. Intraoral excision of large submental dermoid

    Directory of Open Access Journals (Sweden)

    Ankur Bhatnagar

    2013-01-01

    Full Text Available Sublingual dermoids are the rarest forms of craniofacial dermoids mostly seen in young individuals. Excision of large and deep submental dermoid is generally done via extraoral approach scarring the most prominent part of the face, which can lead to post operative scar hypertrophy and hyperpigmentation especially in non-Caucasian races. Presence of such scars leads to adverse psychological effects in young individuals. Excision via intraoral route, although technically demanding, can be simplified using basic principles of plastic surgery leading to optimal aesthetic outcome with least downtime. We excised a large sublingual dermoid extending deep to the mylohyoid muscle through intraoral approach with excellent cosmetic results. Clinicians dealing with such lesions should keep these principals in their armamentarium when dealing with this rare subset of cases.

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

    KAUST Repository

    He, Xinjian

    2011-02-15

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

  11. Applications of nanoparticles for DNA based rabies vaccine.

    Science.gov (United States)

    Shah, Muhammad Ali A; Khan, Sajid Umar; Ali, Zeeshan; Yang, Haowen; Liu, Keke; Mao, Lanlan

    2014-01-01

    Rabies is a fatal encephalomyelitis. Most cases occur in developing countries and are transmitted by dogs. The cell culture vaccines as associated with high cost; therefore, have not replaced the unsafe brain-derived vaccines. In the developing countries these brain-derived rabies vaccines still can be seen in action. Moreover, there will be a need for vaccines against rabies-related viruses against which classical vaccines are not always effective. The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for alternate control strategies. DNA vaccines have emerged as the safest vaccines and best remedy for complicated diseases like hepatitis, HIV, and rabies. A number of recombinant DNA vaccines are now being developed against several diseases such as AIDS and malaria. Therefore, it can be a valuable alternative for the production of cheaper rabies vaccines against its larger spectrum of viruses. In this review we report published data on DNA-based immunization with sequences encoding rabies with special reference to nanotechnology.

  12. Genomic island excisions in Bordetella petrii

    Directory of Open Access Journals (Sweden)

    Levillain Erwan

    2009-07-01

    Full Text Available Abstract Background Among the members of the genus Bordetella B. petrii is unique, since it is the only species isolated from the environment, while the pathogenic Bordetellae are obligately associated with host organisms. Another feature distinguishing B. petrii from the other sequenced Bordetellae is the presence of a large number of mobile genetic elements including several large genomic regions with typical characteristics of genomic islands collectively known as integrative and conjugative elements (ICEs. These elements mainly encode accessory metabolic factors enabling this bacterium to grow on a large repertoire of aromatic compounds. Results During in vitro culture of Bordetella petrii colony variants appear frequently. We show that this variability can be attributed to the presence of a large number of metastable mobile genetic elements on its chromosome. In fact, the genome sequence of B. petrii revealed the presence of at least seven large genomic islands mostly encoding accessory metabolic functions involved in the degradation of aromatic compounds and detoxification of heavy metals. Four of these islands (termed GI1 to GI3 and GI6 are highly related to ICEclc of Pseudomonas knackmussii sp. strain B13. Here we present first data about the molecular characterization of these islands. We defined the exact borders of each island and we show that during standard culture of the bacteria these islands get excised from the chromosome. For all but one of these islands (GI5 we could detect circular intermediates. For the clc-like elements GI1 to GI3 of B. petrii we provide evidence that tandem insertion of these islands which all encode highly related integrases and attachment sites may also lead to incorporation of genomic DNA which originally was not part of the island and to the formation of huge composite islands. By integration of a tetracycline resistance cassette into GI3 we found this island to be rather unstable and to be lost from

  13. Alkylation damage in DNA and RNA--repair mechanisms and medical significance

    DEFF Research Database (Denmark)

    Drabløs, Finn; Feyzi, Emadoldin; Aas, Per Arne

    2004-01-01

    Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions...... are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase......, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents...

  14. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    Directory of Open Access Journals (Sweden)

    Sukumar Biswas

    2016-01-01

    Full Text Available Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR assay and the other loop-mediated isothermal amplification (LAMP assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS, and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise.

  15. Recognizing a Single Base in an Individual DNA Strand: A Step Toward Nanopore DNA Sequencing**

    Science.gov (United States)

    Ashkenasy, N.; Sánchez-Quesada, J.; Ghadiri, M. R.; Bayley, H.

    2007-01-01

    Functional supramolecular chemistry at the single-molecule level. Single strands of DNA can be captured inside α-hemolysin transmembrane pore protein to form single-species α-HL·DNA pseudorotaxanes. This process can be used to identify a single adenine nucleotide at a specific location on a strand of DNA by the characteristic reductions in the α-HL ion conductance. This study suggests that α-HL-mediated single-molecule DNA sequencing might be fundamentally feasible. PMID:15666419

  16. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors.

    Science.gov (United States)

    Chu, Wen-Yi; Huang, Yu-Feng; Huang, Chun-Chin; Cheng, Yi-Sheng; Huang, Chien-Kang; Oyang, Yen-Jen

    2009-07-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein-DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are essential for correct gene regulation. In this respect, ProteDNA is distinctive since it has been designed to identify sequence-specific binding residues. In order to accommodate users with different application needs, ProteDNA has been designed to operate under two modes, namely, the high-precision mode and the balanced mode. According to the experiments reported in this article, under the high-precision mode, ProteDNA has been able to deliver precision of 82.3%, specificity of 99.3%, sensitivity of 49.8% and accuracy of 96.5%. Meanwhile, under the balanced mode, ProteDNA has been able to deliver precision of 60.8%, specificity of 97.6%, sensitivity of 60.7% and accuracy of 95.4%. ProteDNA is available at the following websites: http://protedna.csbb.ntu.edu.tw/, http://protedna.csie.ntu.edu.tw/, http://bio222.esoe.ntu.edu.tw/ProteDNA/.

  17. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors

    OpenAIRE

    2009-01-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein–DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are esse...

  18. DNA-energetics-based analyses suggest additional genes in prokaryotes

    Indian Academy of Sciences (India)

    Garima Khandelwal; Jalaj Gupta; B Jayaram

    2012-07-01

    We present here a novel methodology for predicting new genes in prokaryotic genomes on the basis of inherent energetics of DNA. Regions of higher thermodynamic stability were identified, which were filtered based on already known annotations to yield a set of potentially new genes. These were then processed for their compatibility with the stereo-chemical properties of proteins and tripeptide frequencies of proteins in Swissprot data, which results in a reliable set of new genes in a genome. Quite surprisingly, the methodology identifies new genes even in well-annotated genomes. Also, the methodology can handle genomes of any GC-content, size and number of annotated genes.

  19. Biomaterial-based Memory Device Development by Conducting Metallic DNA

    Science.gov (United States)

    2013-05-28

    basis of the redshift of UV absorption spectra, we think that the incorporation of metal ions may result in a reduction of the original DNA band gap ...memristor based on the changing of the boundary between the high-resistance and low-resistance layers of titanium dioxide TiO2 and TiO2 -x13. Their...microscope (FESEM, JEOL JSM-6500F ) was used to measure the morphology of patterned substrate. The gap width and length between electrodes are both

  20. A New DNA-based Logical Gate Comes into Being

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Across-disciplinary research team, headed by Prof. FAN Chunhai from the CAS Shanghai Institute of Applied Physics, Prof. HE Lin, a CAS Member, and Prof. ZHANG Zhizhou at the Bio-X Research Center under Shanghai Jiao Tong University (SJTU), succeeded in developing a new type of logical gates by applying the deoxyribozyme (DNAzyme), adding a new brick to the groundwork of a DNA-based computation. The related research results have been reported on the German journal Angew. Chem. Int.Ed., 2006, 45, 1759.

  1. Phylogeny of Korean Opuntia spp. based on multiple DNA regions

    OpenAIRE

    SRIKANTH, KRISHNAMOORTHY; WHANG, SUNG SOO

    2015-01-01

    Although Opuntia species are of high agronomic value in Korea, the taxonomic position of Korean Opuntia species has never been investigated. The taxonomic position of Korean Opuntia spp. Within the tribe Opuntieae was examined based on DNA sequence analysis of matK, trnL-F, atpB-rbcl, and ITS regions. The total amplified sequence length was 2977 bp; only 18 parsimonious informative sites were present, even though they belonged to different species. A phylogenetic tree using both the maximum l...

  2. RNA interference against transcription elongation factor SII does not support its role in transcription-coupled nucleotide excision repair.

    Science.gov (United States)

    Mackinnon-Roy, Christine; Stubbert, Lawton J; McKay, Bruce C

    2011-01-10

    RNA polymerase II is unable to bypass bulky DNA lesions induced by agents like ultraviolet light (UV light) and cisplatin that are located in the template strand of active genes. Arrested polymerases form a stable ternary complex at the site of DNA damage that is thought to pose an impediment to the repair of these lesions. Transcription-coupled nucleotide excision repair (TC-NER) preferentially repairs these DNA lesions through an incompletely defined mechanism. Based on elegant in vitro experiments, it was hypothesized that the transcription elongation factor IIS (TFIIS) may be required to couple transcription to repair by catalyzing the reverse translocation of the arrested polymerase, allowing access of repair proteins to the site of DNA damage. However the role of TFIIS in this repair process has not been tested in vivo. Here, silencing TFIIS using an RNA interference strategy did not affect the ability of cells to recover nascent RNA synthesis following UV exposure or the ability of cells to repair a UV-damaged reporter gene while a similar strategy to decrease the expression Cockayne syndrome group B protein (CSB) resulted in the expected repair defect. Furthermore, RNA interference against TFIIS did not increase the sensitivity of cells to UV light or cisplatin while decreased expression of CSB did. Taken together, these results indicate that TFIIS is not limiting for the repair of transcription-blocking DNA lesions and thus the present work does not support a role for TFIIS in TC-NER.

  3. Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor.

    Science.gov (United States)

    Bu, Nan-Nan; Tang, Chun-Xia; He, Xi-Wen; Yin, Xue-Bo

    2011-07-21

    Tetrahedron-structured DNA (ts-DNA) in combination with a functionalized oligonucleotide was used to develop a "turn-on" biosensor for Hg(2+) ions. The ts-DNA provided an improved sensitivity and was used to block the active sites.

  4. Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment

    KAUST Repository

    Aylagas, Eva

    2016-06-10

    Characterization of biodiversity has been extensively used to confidently monitor and assess environmental status. Yet, visual morphology, traditionally and widely used for species identification in coastal and marine ecosystem communities, is tedious and entails limitations. Metabarcoding coupled with high-throughput sequencing (HTS) represents an alternative to rapidly, accurately, and cost-effectively analyze thousands of environmental samples simultaneously, and this method is increasingly used to characterize the metazoan taxonomic composition of a wide variety of environments. However, a comprehensive study benchmarking visual and metabarcoding-based taxonomic inferences that validates this technique for environmental monitoring is still lacking. Here, we compare taxonomic inferences of benthic macroinvertebrate samples of known taxonomic composition obtained using alternative metabarcoding protocols based on a combination of different DNA sources, barcodes of the mitochondrial cytochrome oxidase I gene and amplification conditions. Our results highlight the influence of the metabarcoding protocol in the obtained taxonomic composition and suggest the better performance of an alternative 313 bp length barcode to the traditionally 658 bp length one used for metazoan metabarcoding. Additionally, we show that a biotic index inferred from the list of macroinvertebrate taxa obtained using DNA-based taxonomic assignments is comparable to that inferred using morphological identification. Thus, our analyses prove metabarcoding valid for environmental status assessment and will contribute to accelerating the implementation of this technique to regular monitoring programs.

  5. Solution-based targeted genomic enrichment for precious DNA samples

    Directory of Open Access Journals (Sweden)

    Shearer Aiden

    2012-05-01

    Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

  6. A kinetic and structural investigation of DNA-Based asymmetric catalysis using first-generation ligands

    NARCIS (Netherlands)

    Rosati, Fiora; Boersma, Arnold J.; Klijn, Jaap E.; Meetsma, Auke; Feringa, Ben L.; Roelfes, Gerard

    2009-01-01

    The recently developed concept of DNA-based asymmetric catalysis involves the transfer of chirality from the DNA double helix in reactions using a noncovalently bound catalyst. To date, two generations of DNA-based catalysts have been reported that differ in the design of the ligand for the metal. H

  7. Regulation of nucleotide excision repair by nuclear lamin b1.

    Directory of Open Access Journals (Sweden)

    Veronika Butin-Israeli

    Full Text Available The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1 in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

  8. Intelligent DNA-based molecular diagnostics using linked genetic markers

    Energy Technology Data Exchange (ETDEWEB)

    Pathak, D.K.; Perlin, M.W.; Hoffman, E.P.

    1994-12-31

    This paper describes a knowledge-based system for molecular diagnostics, and its application to fully automated diagnosis of X-linked genetic disorders. Molecular diagnostic information is used in clinical practice for determining genetic risks, such as carrier determination and prenatal diagnosis. Initially, blood samples are obtained from related individuals, and PCR amplification is performed. Linkage-based molecular diagnosis then entails three data analysis steps. First, for every individual, the alleles (i.e., DNA composition) are determined at specified chromosomal locations. Second, the flow of genetic material among the individuals is established. Third, the probability that a given individual is either a carrier of the disease or affected by the disease is determined. The current practice is to perform each of these three steps manually, which is costly, time consuming, labor-intensive, and error-prone. As such, the knowledge-intensive data analysis and interpretation supersede the actual experimentation effort as the major bottleneck in molecular diagnostics. By examining the human problem solving for the task, we have designed and implemented a prototype knowledge-based system capable of fully automating linkage-based molecular diagnostics in X-linked genetic disorders, including Duchenne Muscular Dystrophy (DMD). Our system uses knowledge-based interpretation of gel electrophoresis images to determine individual DNA marker labels, a constraint satisfaction search for consistent genetic flow among individuals, and a blackboard-style problem solver for risk assessment. We describe the system`s successful diagnosis of DMD carrier and affected individuals from raw clinical data.

  9. Mechanisms by which Human Cells Bypass Damaged Bases during DNA Replication after Ultraviolet Irradiation

    Directory of Open Access Journals (Sweden)

    James E. Cleaver

    2002-01-01

    Full Text Available The replication of damaged DNA involves cascading mechanisms of increasing complexity but decreasing accuracy. The most accurate mechanism uses low-fidelity DNA polymerases, Pol H and Pol I, which have active sites sufficiently large to accommodate a pyrimidine dimer. Replicative bypass of DNA damage by these polymerases produces an accurately replicated, newly synthesized strand. Pol H negative cells (XP-V cell lines either adopt a proposed secondary bypass mechanism or a recombinational mode. The mechanism of the secondary bypass is unclear, but a number of experiments suggests roles for excision repair to remove damage ahead of replication forks, hRad6/18 proteolysis to clear the blocked forks, and the Rad17-RFC and 9-1-1 complexes to establish a new replication apparatus. This alternative pathway requires functional p53. In Pol H negative cells in which p53 is also inactive, the arrested fork fragments into DNA double strand breaks. Foci containing PCNA, Mre11/Rad50/Nbs1, and gamma-H2Ax can then be detected, along with chromosomal rearrangement and high frequencies of sister chromatid exchanges. The recruitment of recombination components to the arrested forks represents the ultimate failure of replication machinery to relieve the arrested state and bypass the damage. The resulting chromosomal instability in surviving cells will contribute to malignant transformation.

  10. DNA microarray-based mutation discovery and genotyping.

    Science.gov (United States)

    Gresham, David

    2011-01-01

    DNA microarrays provide an efficient means of identifying single-nucleotide polymorphisms (SNPs) in DNA samples and characterizing their frequencies in individual and mixed samples. We have studied the parameters that determine the sensitivity of DNA probes to SNPs and found that the melting temperature (T (m)) of the probe is the primary determinant of probe sensitivity. An isothermal-melting temperature DNA microarray design, in which the T (m) of all probes is tightly distributed, can be implemented by varying the length of DNA probes within a single DNA microarray. I describe guidelines for designing isothermal-melting temperature DNA microarrays and protocols for labeling and hybridizing DNA samples to DNA microarrays for SNP discovery, genotyping, and quantitative determination of allele frequencies in mixed samples.

  11. Magnetic particle-based sandwich sensor with DNA-modified carbon nanotubes as recognition elements for detection of DNA hybridization.

    Science.gov (United States)

    Hu, Po; Huang, Cheng Zhi; Li, Yuan Fang; Ling, Jian; Liu, Yu Ling; Fei, Liang Run; Xie, Jian Ping

    2008-03-01

    In this contribution, we design a visual sensor for DNA hybridization with DNA probe-modified magnetic particles (MPs) and multiwalled carbon nanotubes (MWNTs) without involving a visual recognition element such as fluorescent/chemiluminescent reagents. It was found that DNA probe-modified MWNTs, which could be dispersed in aqueous medium and have strong light scattering signals under the excitation of a light beam in the UV-vis region, could connect with DNA probe-modified MPs together in the presence of perfectly complementary target DNA and form a sandwich structure. In a magnetic field, the formed MP-MWNT species can easily be removed from the solution, resulting in a decrease of light scattering signals. Thus, a magnetic particle-based sandwich sensor could be developed to detect DNA hybridization by measuring the light scattering signals with DNA-modified MWNTs as recognition elements. Experiments showed that the DNA-modified MPs sensor could be reused at least 17 times and was stable for more than 6 months.

  12. Keeping Uracil Out of DNA: Physiological Role, Structure and Catalytic Mechanism of dUTPases

    OpenAIRE

    2009-01-01

    The thymine-uracil exchange constitutes one of the major chemical differences between DNA and RNA. Although these two bases form the same Watson-Crick base pairs with adenine and are equivalent for both information storage and transmission, uracil incorporation in DNA is usually a mistake that needs to be excised. There are two ways for uracil to appear in DNA: thymine replacement and cytosine deamination. Most DNA polymerases readily incorporate dUMP as well as dTMP depending solely on the a...

  13. Ultrafast dynamics in DNA base pairs following ultraviolet excitation.

    Science.gov (United States)

    Orr-Ewing, Andrew

    2015-03-01

    Photo-protective mechanisms in DNA are essential to maintain the integrity of the genetic code by preventing damage from absorption of solar ultraviolet (UV) radiation. We have used time-resolved infra-red (TRIR) spectroscopy to observe the dynamics of Watson-Crick nucleobase pairs following absorption of femtosecond UV laser pulses. The base pairs are prepared as nucleosides in solution, and photo-induced dynamics are probed in the carbonyl and N-H bond stretching regions using broadband IR pulses with picosecond time resolution. Results will be presented for the guanine-cytosine (G-C) base pair, contrasting the rapid recovery of ground-state products (the photo-protection pathway) with formation of other photoproducts which might represent photo-damage mechanisms. This work is a collaboration with the group of Prof F. Temps (Christian-Albrechts-Universitat zu Kiel). This research is supported by ERC Advanced Grant 290966 CAPRI.

  14. DNA repair. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, R.

    1978-01-01

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

  15. DNA modifications in atherosclerosis: from the past to the future.

    Science.gov (United States)

    Borghini, Andrea; Cervelli, Tiziana; Galli, Alvaro; Andreassi, Maria Grazia

    2013-10-01

    The role of DNA damage in the pathogenesis of atherosclerosis has been extensively investigated in recent decades. There is now clear that oxidative stress is an important inducer of both DNA damage and telomere attrition which, in turn, can gives rise to genome instability and vascular senescence. This review discusses the role of the DNA damage response, including the key DNA repair pathways (base excision repair, nucleotide excision repair, homologous recombination and non-homologous end joining), deregulated cell cycle and apoptosis in atherosclerosis. We also highlight emerging evidence suggesting that epigenetic changes (DNA methylation and microRNA-mediated mechanisms), not associated with alterations in DNA sequences, may play a critical role in the regulation of the DNA damage response. Nevertheless, further investigation is still required to better understand the complexity of DNA repair and DNA damage response in atherosclerosis, making this topic an exciting and promising field for future investigation. Unraveling these molecular mechanisms provide the rationale for the development of novel efficient therapies to combat the vascular aging process.

  16. Recognizing a Single Base in an Individual DNA Strand: A Step Toward Nanopore DNA Sequencing**

    OpenAIRE

    Ashkenasy, N.; Sánchez-Quesada, J.; Ghadiri, M. R.; Bayley, H

    2005-01-01

    Functional supramolecular chemistry at the single-molecule level. Single strands of DNA can be captured inside α-hemolysin transmembrane pore protein to form single-species α-HL·DNA pseudorotaxanes. This process can be used to identify a single adenine nucleotide at a specific location on a strand of DNA by the characteristic reductions in the α-HL ion conductance. This study sug...

  17. DNA barcoding: error rates based on comprehensive sampling.

    Directory of Open Access Journals (Sweden)

    Christopher P Meyer

    2005-12-01

    Full Text Available DNA barcoding has attracted attention with promises to aid in species identification and discovery; however, few well-sampled datasets are available to test its performance. We provide the first examination of barcoding performance in a comprehensively sampled, diverse group (cypraeid marine gastropods, or cowries. We utilize previous methods for testing performance and employ a novel phylogenetic approach to calculate intraspecific variation and interspecific divergence. Error rates are estimated for (1 identifying samples against a well-characterized phylogeny, and (2 assisting in species discovery for partially known groups. We find that the lowest overall error for species identification is 4%. In contrast, barcoding performs poorly in incompletely sampled groups. Here, species delineation relies on the use of thresholds, set to differentiate between intraspecific variation and interspecific divergence. Whereas proponents envision a "barcoding gap" between the two, we find substantial overlap, leading to minimal error rates of approximately 17% in cowries. Moreover, error rates double if only traditionally recognized species are analyzed. Thus, DNA barcoding holds promise for identification in taxonomically well-understood and thoroughly sampled clades. However, the use of thresholds does not bode well for delineating closely related species in taxonomically understudied groups. The promise of barcoding will be realized only if based on solid taxonomic foundations.

  18. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    Science.gov (United States)

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  19. Quality Control of Isothermal Amplified DNA Based on Short Tandem Repeat Analysis.

    Science.gov (United States)

    Kroneis, Thomas; El-Heliebi, Amin

    2015-01-01

    This protocol describes the use of a 16plex PCR for the purpose assessing DNA quality after isothermal whole genome amplification (WGA). In short, DNA products, generated by amplification multiple displacement amplification, are forwarded to PCR targeting 15 short tandem repeats (STR) as well as amelogenin generating up to 32 different PCR products. After amplification, the PCR products are separated via capillary electrophoresis and analyzed based on the obtained DNA profiles. Isothermal WGA products of good DNA quality will result in DNA profiles with efficiencies of >90 % of the full DNA profile.

  20. DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.

    Science.gov (United States)

    Ross, H A; Lento, G M; Dalebout, M L; Goode, M; Ewing, G; McLaren, P; Rodrigo, A G; Lavery, S; Baker, C S

    2003-01-01

    DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.

  1. The prognostic and predictive value of excision repair cross-complementation group 1 (ERCC1) protein in 1288 patients with head and neck squamous cell carcinoma treated with platinum-based therapy: a meta-analysis.

    Science.gov (United States)

    Bišof, Vesna; Zajc Petranović, Matea; Rakušić, Zoran; Samardžić, Kristina Ruža; Juretić, Antonio

    2016-09-01

    Excision repair cross-complementation group 1 (ERCC1) protein has been extensively investigated as a prognostic and predictive factor for platinum-based treatment in head and neck squamous cell carcinoma (HNSCC) but with inconsistent results. We performed the present meta-analysis to better elucidate this issue in advanced HNSCC. A literature search was conducted using the PubMed and Web of Science databases. The inclusion criteria were head and neck cancer patients with platinum-based treatment and evaluation of the correlation between ERCC1 expression and clinical outcomes [objective response rate (ORR), progression-free survival (PFS), and overall survival (OS), both unadjusted and adjusted estimates]. In high vs. low pooled analyses, high ERCC1 expression was associated with unfavorable OS [hazard ratio (HR) = 1.95, 95 % confidence interval (CI) 1.18-3.21, p = 0.009], PFS (HR = 2.39, 95 % CI 1.74-3.28, p = 0.000) and ORR (odds ratio = 0.48, 95 % CI 0.23-0.98, p = 0.044). In the subgroup analysis of adjusted OS estimates, ERCC1 was a predictor of shorter survival in Asians (HR = 3.13, 95 % CI 2.09-4.70, p = 0.000) and Caucasians (HR = 2.02, 95 % CI 1.32-3.07, p = 0.001) but of longer survival in South Americans (HR = 0.17, 95 % CI 0.07-0.40, p = 0.000). Immunohistochemistry proved to be of predictive value irrespective of used antibody (p = 0.009). In the stratified analysis according to the tumor site, ERCC1 expression was associated with OS in nasopharyngeal cancer (HR = 2.72, 95 % CI 1.79-4.13, p = 0.000). ERCC1 has a potential to become predictive and prognostic factor enabling treatment tailoring in HNSCC patients.

  2. Roles of the Amino Group of Purine Bases in the Thermodynamic Stability of DNA Base Pairing

    Directory of Open Access Journals (Sweden)

    Shu-ichi Nakano

    2014-08-01

    Full Text Available The energetic aspects of hydrogen-bonded base-pair interactions are important for the design of functional nucleotide analogs and for practical applications of oligonucleotides. The present study investigated the contribution of the 2-amino group of DNA purine bases to the thermodynamic stability of oligonucleotide duplexes under different salt and solvent conditions, using 2'-deoxyriboinosine (I and 2'-deoxyribo-2,6-diaminopurine (D as non-canonical nucleotides. The stability of DNA duplexes was changed by substitution of a single base pair in the following order: G•C > D•T ≈ I•C > A•T > G•T > I•T. The apparent stabilization energy due to the presence of the 2-amino group of G and D varied depending on the salt concentration, and decreased in the water-ethanol mixed solvent. The effects of salt concentration on the thermodynamics of DNA duplexes were found to be partially sequence-dependent, and the 2-amino group of the purine bases might have an influence on the binding of ions to DNA through the formation of a stable base-paired structure. Our results also showed that physiological salt conditions were energetically favorable for complementary base recognition, and conversely, low salt concentration media and ethanol-containing solvents were effective for low stringency oligonucleotide hybridization, in the context of conditions employed in this study.

  3. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    Directory of Open Access Journals (Sweden)

    Hiroshi Arakawa

    2015-06-01

    Full Text Available Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1, DNA ligase 3 (Lig3 and DNA ligase 4 (Lig4. While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER, homologous recombination repair (HRR and nucleotide excision repair (NER. Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ. Lig3 is implicated in a short-patch base excision repair (BER pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche

  4. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    Science.gov (United States)

    Arakawa, Hiroshi; Iliakis, George

    2015-06-23

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

  5. Pros and cons of methylation-based enrichment methods for ancient DNA

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio

    2015-01-01

    The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules contai...

  6. Development of a lipase-based optical assay for detection of DNA

    DEFF Research Database (Denmark)

    Pinijsuwan, Suttiporn; Shipovskov, Stepan; Surareungchai, Werasak

    2011-01-01

    A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate...

  7. Phylogeny of Pelargonium (Geraniaceae) based on DNA sequences from three genomes

    NARCIS (Netherlands)

    Bakker, F.T.; Culham, A.; Hettiarachi, P.; Touloumendidou, T.; Gibby, M.

    2004-01-01

    Phylogenetic hypotheses for the largely South African genus Pelargonium L'Hér. (Geraniaceae) were derived based on DNA sequence data from nuclear, chloroplast and mitochondrial encoded regions. The datasets were unequally represented and comprised cpDNA trnL-F sequences for 152 taxa, nrDNA ITS seque

  8. Conservation and divergence of DNA methylation in eukaryotes: new insights from single base-resolution DNA methylomes.

    Science.gov (United States)

    Su, Zhixi; Han, Leng; Zhao, Zhongming

    2011-02-01

    DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at single-base resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.

  9. PCR-based detection of a rare linear DNA in cell culture

    Directory of Open Access Journals (Sweden)

    Saveliev Sergei V.

    2002-01-01

    Full Text Available The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

  10. Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe.

    Science.gov (United States)

    Fogg, Paul C M; Haley, Joshua A; Stark, W Marshall; Smith, Margaret C M

    2017-03-01

    Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox.IMPORTANCEStreptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different

  11. Nucleotide excision repair is associated with the replisome and its efficiency depends on a direct interaction between XPA and PCNA.

    Directory of Open Access Journals (Sweden)

    Karin M Gilljam

    Full Text Available Proliferating cell nuclear antigen (PCNA is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM. The xeroderma pigmentosum group A (XPA protein, with a central but somewhat elusive role in nucleotide excision repair (NER, contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA(-/- cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6-4 photoproducts, and are consequently more arrested in S phase as compared to XPA(-/- cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.

  12. 75 FR 9359 - Drawback of Internal Revenue Excise Tax

    Science.gov (United States)

    2010-03-02

    ... Parts 113 and 191 RIN 1505-AC18 Drawback of Internal Revenue Excise Tax AGENCY: Customs and Border... Regulations to: preclude the filing of a substitution drawback claim for internal revenue excise tax paid on imported merchandise in situations where no excise tax was paid upon the substituted merchandise or...

  13. Direct electrochemical detection of PCR product based on charge transfer through DNA

    Institute of Scientific and Technical Information of China (English)

    ZHAO Hongtao; ZHANG Zhijie; JU Huangxian

    2005-01-01

    @@ Human genome project and genetic identification for inherited diseases will definitely have a profound impact on the diagnosis of diseases[1], which calls for rapid and accurate assays of DNA. Among different types of sensors, electrochemical DNA biosensors offer a promising alternative means[2,3]. Recent efforts to elucidate the mechanism of charge transfer in DNA have demonstrated that the charge transfer is sensitive to the perturbation in base stack[4,5]. Long-range charge transfer in DNA therefore has been showing great potential application in the development of DNA-based biosensors, especially in the study of single nucleotide polymorphs[7―10].

  14. D2 gastrectomy and complete mesentery excision based on metastasis Ⅴ and membrane anatomy%从“膜解剖”和“第五转移”看胃癌根治术的规范化实施

    Institute of Scientific and Technical Information of China (English)

    龚建平

    2015-01-01

    D2 procedure, which includes dissection of the lymph nodes and adipose tissue in the field, has been widely accepted as a standard for curative gastric cancer surgery. However, there is no description for the boundary of these adipose resection in D2 procedure protocol so far. We found that there was a membranous tissue plane which enveloped the stomach, blood vessels, lymphatic and adipose together, and suspended them to the posterior wall of abdomen. This structure is consistent with the definition of the mesentery and we call it the mesogastrium, indicating a metastasis V in it. The precise D2 procedure should include the complete mesentery excision based on membrane anatomy, which may lead to less bleeding, more complete dissemination.%D2手术作为胃癌根治的标准术式已被广泛接受,这一手术强调胃周一定范围内的淋巴结清扫和脂肪结缔组织的切除。但这些脂肪结缔组织切除的边界尚未给予界定。我们研究发现,有一层膜包绕着胃及其周围血管、淋巴和脂肪结缔组织,并将其悬挂于后腹壁。由于这种结构符合系膜的定义,我们称其为胃系膜。在胃系膜内不仅有淋巴结转移存在,还可能有“第五转移”。因此我们倡导,在行传统D2手术的同时,尽可能在膜解剖的基础上完整地切除胃系膜,以便规范D2手术并推广之。

  15. [Local excision of giant rectal polypoid neoplasms].

    Science.gov (United States)

    Cimitan, Andrea; Burza, Antonio; Basile, Ursula; Saputo, Serena; Mingazzini, Pietro; Stipa, Francesco

    2008-01-01

    Local excision is the best therapeutic option for giant adenomas of the rectum. Parks technique for lower rectal lesions and the T.E.M. technique for lesions localised in the middle and upper rectum offer exceptionally good exposure, allowing radical excision in the case of early low-risk T1 adenocarcinomas (well or moderately differentiated [G1/2] without lymphovascular invasion [L0]). From July 1987 to March 2006, 224 patients were treated by local excision for rectal lesions in our department. In 48 patients (21.4%) a large sessile benign lesion was diagnosed preoperatively. In 3 patients with a preoperative diagnosis of severe dysplasia (Tis) final pathology showed adenoma and for this reason they were included in our study group. A total of 51 patients with giant preoperative benign lesions were treated by local excision (Parks technique, T.E.M. or both). Twenty-five (49%) patients had a definitive diagnosis of adenocarcinoma: in situ (pTis) in 22 patients (88%), pT1 in 2 patients (8%) and pT2 in 1 patient (4%). In 26 patients (51%) the diagnosis was adenoma. The overall local recurrence rate was 9.8% (5/51); the recurrence rate was 7.6% (2/26) for adenomas and 12% (3/25) for carcinomas. The median hospital stay was 7 days (range 3-39). There was no operative mortality. Giant sessile polypoid lesions localized in the middle and upper rectum are best treated with T.E.M., while Parks technique is a good option in lower rectal tumours. These techniques, if correctly indicated and well performed, offer great advantages in terms of safety and radicality. In our experience the operative mortality was nil and the morbidity and recurrence rates were low.

  16. The 2015 Nobel Prize in Chemistry The Discovery of Essential Mechanisms that Repair DNA Damage.

    Science.gov (United States)

    Lindahl, Tomas; Modrich, Paul; Sancar, Aziz

    2016-01-01

    The Royal Swedish Academy awarded the Nobel Prize in Chemistry for 2015 to Tomas Lindahl, Paul Modrich and Aziz Sancar for their discoveries in fundamental mechanisms of DNA repair. This pioneering research described three different essential pathways that correct DNA damage, safeguard the integrity of the genetic code to ensure its accurate replication through generations, and allow proper cell division. Working independently of each other, Tomas Lindahl, Paul Modrich and Aziz Sancar delineated the mechanisms of base excision repair, mismatch repair and nucleotide excision repair, respectively. These breakthroughs challenged and dismissed the early view that the DNA molecule was very stable, paving the way for the discovery of human hereditary diseases associated with distinct DNA repair deficiencies and a susceptibility to cancer. It also brought a deeper understanding of cancer as well as neurodegenerative or neurological diseases, and let to novel strategies to treat cancer.

  17. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    Science.gov (United States)

    Gardner, Shea N.; Mariella, Jr., Raymond P.; Christian, Allen T.; Young, Jennifer A.; Clague, David S.

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  18. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability

    Science.gov (United States)

    Sollier, Julie; Stork, Caroline Townsend; García-Rubio, María L.; Paulsen, Renee D.; Aguilera, Andrés; Cimprich, Karlene A.

    2014-01-01

    Summary R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability, however the mechanisms underlying R-loop induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability. PMID:25435140

  19. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability.

    Science.gov (United States)

    Sollier, Julie; Stork, Caroline Townsend; García-Rubio, María L; Paulsen, Renee D; Aguilera, Andrés; Cimprich, Karlene A

    2014-12-18

    R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

  20. DNA repair in Chromobacterium violaceum.

    Science.gov (United States)

    Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

    2004-03-31

    Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

  1. Repair of 8-oxodeoxyguanosine lesions in mitochondrial DNA depends on the oxoguanine DNA glycosylase (OGG1) gene and 8- oxoguanine accumulates in the mitochondrial DNA of OGG1- defective mice

    DEFF Research Database (Denmark)

    Souza-Pinto, N.C.; Eide, L.; Hogue, B.A.

    2001-01-01

    encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision activity toward an oligonucleotide containing a single 8-oxodG DNA base lesion, Consistent with an important role for the OGG1 protein in the removal of 8-oxodC from the mitochondrial genome, we found that mtDNA......Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodC), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear...... DNA, The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1), We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8...

  2. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

    Science.gov (United States)

    Gryson, Nicolas

    2010-03-01

    The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

  3. DNA purification and gene typing:Based on multifunctional nanobeads

    Institute of Scientific and Technical Information of China (English)

    XIE Xin; ZHANG Xu; GAO Huafang; ZHANG Huan; CHEN Depu; CHENG Jing; FEI Weiyang

    2004-01-01

    In this report, a universal protocol for extracting genomic DNA from whole blood, saliva, and bacterial culture by using magnetic nanobeads as solid-phase absorbents was presented. The enrichment of target cells and adsorption of DNA have been functionally integrated onto the surfaces of the carboxyl-modified magnetic nano-beads, and the DNA segments bound on the surface of the beads can be directly used as PCR templates to amplify a target gene. The PCR products were applied to an oligonucleotide array to perform gene typing. The protocol proves to be simple, rapid, biologically and chemically nonhazardous, and promising for the microfabrication of DNA preparation chip.

  4. Job shop scheduling problem based on DNA computing

    Institute of Scientific and Technical Information of China (English)

    Yin Zhixiang; Cui Jianzhong; Yang Yan; Ma Ying

    2006-01-01

    To solve job shop scheduling problem, a new approach-DNA computing is used in solving job shop scheduling problem. The approach using DNA computing to solve job shop scheduling is divided into three stands. Finally, optimum solutions are obtained by sequencing. A small job shop scheduling problem is solved in DNA computing, and the "operations" of the computation were performed with standard protocols, as ligation, synthesis, electrophoresis etc. This work represents further evidence for the ability of DNA computing to solve NP-complete search problems.

  5. DBD-Hunter: a knowledge-based method for the prediction of DNA-protein interactions.

    Science.gov (United States)

    Gao, Mu; Skolnick, Jeffrey

    2008-07-01

    The structures of DNA-protein complexes have illuminated the diversity of DNA-protein binding mechanisms shown by different protein families. This lack of generality could pose a great challenge for predicting DNA-protein interactions. To address this issue, we have developed a knowledge-based method, DNA-binding Domain Hunter (DBD-Hunter), for identifying DNA-binding proteins and associated binding sites. The method combines structural comparison and the evaluation of a statistical potential, which we derive to describe interactions between DNA base pairs and protein residues. We demonstrate that DBD-Hunter is an accurate method for predicting DNA-binding function of proteins, and that DNA-binding protein residues can be reliably inferred from the corresponding templates if identified. In benchmark tests on approximately 4000 proteins, our method achieved an accuracy of 98% and a precision of 84%, which significantly outperforms three previous methods. We further validate the method on DNA-binding protein structures determined in DNA-free (apo) state. We show that the accuracy of our method is only slightly affected on apo-structures compared to the performance on holo-structures cocrystallized with DNA. Finally, we apply the method to approximately 1700 structural genomics targets and predict that 37 targets with previously unknown function are likely to be DNA-binding proteins. DBD-Hunter is freely available at http://cssb.biology.gatech.edu/skolnick/webservice/DBD-Hunter/.

  6. Stretched and overwound DNA forms a Pauling-like structure with exposed bases.

    Science.gov (United States)

    Allemand, J F; Bensimon, D; Lavery, R; Croquette, V

    1998-11-24

    We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from sigma approximately -0.015 down to sigma = -1. Surprisingly, for positively supercoiled DNA (sigma > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has approximately 2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling's early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84-97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671-674].

  7. Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation.

    Science.gov (United States)

    Dingler, Felix A; Kemmerich, Kristin; Neuberger, Michael S; Rada, Cristina

    2014-07-01

    Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil-DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung(-/-) mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung(-/-) mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung(-/-) mice depends upon the endogenous SMUG1 uracil-DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung(-/-) Smug1(-/-) mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2-dependent mechanism, Ung(-/-) Smug1(-/-) mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung(-/-) background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.

  8. Development of a DNA Sensor Based on Alkanethiol Self- Assembled Monolayer-Modified Electrodes

    Directory of Open Access Journals (Sweden)

    José M. Pingarrón

    2005-11-01

    Full Text Available An electrochemical DNA biosensor based on recognition of double or singlestranded DNA (ds-DNA/ss-DNA immobilised on a self-assembled modified gold electrodeis presented for denaturalisation and hybridisation detection. DNA is covalently bond on aself assembled 3-mercaptopropionic acid monolayer by using water soluble N-3-(dimethylaminopropyl-N´ethylcarbodiimide hydrochloride (EDC and Nhydroxisulfosuccinimide(NHSS as linkers. The interaction between the immobilised DNAand methylene blue (MB is investigated using square wave voltammetry (SWV. Theincrease or diminution of peak currents of the MB upon the hybridisation or denaturalisationevent at the modified electrode surface is studied.

  9. Biomarkers of oxidative damage to DNA and repair

    DEFF Research Database (Denmark)

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

    2008-01-01

    , which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7......,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine...

  10. Molecular Design of Ionization-Induced Proton Switching Element Based on Fluorinated DNA Base Pair.

    Science.gov (United States)

    Tachikawa, Hiroto; Kawabata, Hiroshi

    2016-03-10

    To design theoretically the high-performance proton switching element based on DNA base pair, the effects of fluorine substitution on the rate of proton transfer (PT) in the DNA model base pair have been investigated by means of direct ab initio molecular dynamics (AIMD) method. The 2-aminopyridine dimer, (AP)2, was used as the model of the DNA base pair. One of the hydrogen atoms of the AP molecule in the dimer was substituted by a fluorine (F) atom, and the structures of the dimer, expressed by F-(AP)2, were fully optimized at the MP2/6-311++G(d,p) level. The direct AIMD calculations showed that the proton is transferred within the base pair after the vertical ionization. The rates of PT in F-(AP)2(+) were calculated and compared with that of (AP)2(+) without an F atom. It was found that PT rate is accelerated by the F-substitution. Also, the direction of PT between F-AP and AP molecules can be clearly controlled by the position of F-substitution (AP)2 in the dimer.

  11. Potential for DNA-based identification of Great Lakes fauna: Match and mismatch between taxa inventories and DNA barcode libraries

    Science.gov (United States)

    DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to biotic condition assessment and non-native species early-detection monitoring. However, the abi...

  12. New insights on the mechanism of quinoline-based DNA Methyltransferase inhibitors.

    Science.gov (United States)

    Gros, Christina; Fleury, Laurence; Nahoum, Virginie; Faux, Céline; Valente, Sergio; Labella, Donatella; Cantagrel, Frédéric; Rilova, Elodie; Bouhlel, Mohamed Amine; David-Cordonnier, Marie-Hélène; Dufau, Isabelle; Ausseil, Frédéric; Mai, Antonello; Mourey, Lionel; Lacroix, Laurent; Arimondo, Paola B

    2015-03-06

    Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.

  13. Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry.

    Science.gov (United States)

    Wu, Nai-ying; Gao, Wei; He, Xu-lun; Chang, Zhu; Xu, Mao-tian

    2013-01-15

    A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.

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

    Directory of Open Access Journals (Sweden)

    Elisa Mentegari

    2016-08-01

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

  15. Synthetic lethal targeting of DNA double strand break repair deficient cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors

    OpenAIRE

    Sultana, Rebeka; McNeill, Daniel R.; Abbotts, Rachel; Mohammed, Mohammed Z.; Zdzienicka, Małgorzata Z.; Qutob, Haitham; Seedhouse, Claire; Charles A. Laughton; Fischer, Peter M.; Patel, Poulam M.; Wilson, David M.; Madhusudan, Srinivasan

    2012-01-01

    An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chine...

  16. Ultratrace DNA Detection Based on the Condensing-Enrichment Effect of Superwettable Microchips.

    Science.gov (United States)

    Xu, Li-Ping; Chen, Yanxia; Yang, Gao; Shi, Wanxin; Dai, Bing; Li, Guannan; Cao, Yanhua; Wen, Yongqiang; Zhang, Xueji; Wang, Shutao

    2015-11-18

    A sensitive nucleic acid detection platform based on superhydrophilic microwells spotted on a superhydrophobic substrate is fabricated. Due to the wettability differences, ultratrace DNA molecules are enriched and the fluorescent signals are amplified to allow more sensitive detection. The biosensing interface based on superwettable materials provides a simple and cost-effective way for ultratrace DNA sensing.

  17. High Interlaboratory Reprocucibility of DNA Sequence-based Typing of Bacteria in a Multicenter Study

    DEFF Research Database (Denmark)

    Sousa, MA de; Boye, Kit; Lencastre, H de;

    2006-01-01

    Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...

  18. Microcantilver-based DNA hybridization sensors for Salmonella identification

    Directory of Open Access Journals (Sweden)

    Carlo Ricciardi

    2012-02-01

    Full Text Available The detection of pathogenic microorganisms in foods remains a challenging since the safety of foodstuffs has to be ensured by the food producing companies. Conventional methods for the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. Biomolecular methods, are commonly used as a support for traditional techniques, thanks to their high sensitivity, specificity and not excessive costs. However, new methods like biosensors for example, can be an exciting alternative to the more traditional tecniques for the detection of pathogens in food. In this study we report Salmonella enterica serotype Enteritidis DNA detection through a novel class of label-free biosensors: microcantilevers (MCs. In general, MCs can operate as a microbalance and is used to detect the mass of the entities anchored to the cantilever surface using the decrease in the resonant frequency. We use DNA hybridization as model reaction system and for this reason, specific single stranded probe DNA of the pathogen and three different DNA targets (single-stranded complementary DNA, PCR product and serial dilutions of DNA extracted from S. Enteritidis strains were applied. Two protocols were reported in order to allow the probe immobilization on cantilever surface: i MC surface was functionalized with 3-aminopropyltriethoxysilane and glutaraldehyde and an amino-modified DNA probe was used; ii gold-coated sensors and thiolated DNA probes were used in order to generate a covalent bonding (Th-Au. For the first one, measures after hybridization with the PCR product showed related frequency shift 10 times higher than hybridization with complementary probe and detectable signals were obtained at the concentrations of 103 and 106 cfu/mL after hybridization with bacterial DNA. There are currently optimizations of the second protocol, where preliminary results have shown to be more uniform and therefore more precise within each of the

  19. DNA Ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair

    Science.gov (United States)

    Gao, Yankun; Katyal, Sachin; Lee, Youngsoo; Zhao, Jingfeng; Rehg, Jerold E.; Russell, Helen R.; McKinnon, Peter J.

    2011-01-01

    DNA replication and repair in mammalian cells involves three distinct DNA ligases; ligase I (Lig1), ligase III (Lig3) and ligase IV (Lig4)1. Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway2,3. Lig3 is also present in the mitochondria where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc14. However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality5. Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss6, further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair. PMID:21390131

  20. DNA gridiron nanostructures based on four-arm junctions.

    Science.gov (United States)

    Han, Dongran; Pal, Suchetan; Yang, Yang; Jiang, Shuoxing; Nangreave, Jeanette; Liu, Yan; Yan, Hao

    2013-03-22

    Engineering wireframe architectures and scaffolds of increasing complexity is one of the important challenges in nanotechnology. We present a design strategy to create gridiron-like DNA structures. A series of four-arm junctions are used as vertices within a network of double-helical DNA fragments. Deliberate distortion of the junctions from their most relaxed conformations ensures that a scaffold strand can traverse through individual vertices in multiple directions. DNA gridirons were assembled, ranging from two-dimensional arrays with reconfigurability to multilayer and three-dimensional structures and curved objects.

  1. Solid phase based DNA solution of the coloring problem

    Institute of Scientific and Technical Information of China (English)

    PAN Linqiang; LIU Guangwu; XU Jin; LIU Yachun

    2004-01-01

    DNA computing has the potential to tackle computationally difficult problems that have real-world implications.The parallel search capabilities of DNA make it a valuable tool for approaching intractable computational problems,for which conventional computers have limited potentials.Up to now,many accomplishments have been achieved to improve its performance and increase its reliability.In this paper,the coloring problem has been solved by means of molecular biology techniques.The coloring problem is a well-known NP-complete problem.This work represents further evidence for the ability of DNA computing to solve NP-complete problems.

  2. DNA-based materials and their device applications (Conference Presentation)

    Science.gov (United States)

    Rau, Ileana; Kajzar, François; Grote, James G.

    2016-10-01

    In the last decade a lot of interest was paid to DNA materials in view of their practical applications in photonics and in electronics. This aspect is especially due to the fact that this polymer is eco-friendly, originating from renewable resources and can be obtained from any animal or vegetable waste. In this respect many studies have shown that DNA is an intriguing biopolymer which can find applications in many fields. In this paper we will review and discuss the functionalization of DNA and some practical applications.

  3. An electrochemical DNA biosensor based on Oracet Blue as a label for detection of Helicobacter pylori.

    Science.gov (United States)

    Hajihosseini, Saeedeh; Nasirizadeh, Navid; Hejazi, Mohammad Saeid; Yaghmaei, Parichereh

    2016-10-01

    An innovative method of a DNA electrochemical biosensor based on Oracet Blue (OB) as an electroactive label and gold electrode (AuE) for detection of Helicobacter pylori, was offered. A single-stranded DNA probe with a thiol modification was covalently immobilized on the surface of the AuE by forming an Au-S bond. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of reduction of the OB binding to double-stranded DNA (ds-DNA). Our results showed that OB-based DNA biosensor has a decent potential for detection of single-base mismatch in target DNA. Selectivity of the proposed DNA biosensor was further confirmed in the presence of non-complementary and complementary DNA strands. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 0.3nmolL(-1) to 240.0nmolL(-1), and the detection limit was 0.17nmolL(-1), whit a promising reproducibility and repeatability.

  4. Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor.

    Directory of Open Access Journals (Sweden)

    Varun Rai

    Full Text Available A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12 M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.

  5. Silica-Based Solid Phase Extraction of DNA on a Microchip

    Institute of Scientific and Technical Information of China (English)

    陈晓芳; 沈科跃; 刘鹏; 郭旻; 程京; 周玉祥

    2004-01-01

    Micro total analysis systems for chemical and biological analysis have attracted much attention.However,microchips for sample preparation and especially DNA purification are still underdeveloped.This work describes a solid phase extraction chip for purifying DNA from biological samples based on the adsorption of DNA on bare silica beads prepacked in a microchannel.The chip was fabricated with poly-dimethylsiloxane.The silica beads were packed in the channel on the chip with a tapered microchannel to form the packed bed.Fluorescence detection was used to evaluate the DNA adsorbing efficiency of the solid phase.The polymerase chain reaction was used to evaluate the quality of the purified DNA for further use.The extraction efficiency for the DNA extraction chip is approximately 50% with a 150-nL extraction volume.Successful amplification of DNA extracted from human whole blood indicates that this method is compatible with the polymerase chain reaction.

  6. The Emerging Roles of ATP-Dependent Chromatin Remodeling Enzymes in Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Wioletta Czaja

    2012-09-01

    Full Text Available DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo.

  7. Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair.

    Science.gov (United States)

    Lin, Yunfu; Wilson, John H

    2007-09-01

    Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replication. We showed previously that transcription through CAG repeats induces their instability (Y. Lin, V. Dion, and J. H. Wilson, Nat. Struct. Mol. Biol. 13:179-180). Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that short interfering RNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. In contrast, knockdowns of OGG1 and APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcription factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a transcription block. Repeats also are stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway for transcription-induced CAG repeat instability that can account for the contractions observed here and potentially for the contractions and expansions seen with human diseases.

  8. Modulated Lapped Biorthogonal Transform for Non-orthogonal Narrowband Interference Excision in Spread Spectrum Communications

    Institute of Scientific and Technical Information of China (English)

    朱丽平; 胡光锐; 赵海波; 朱义胜

    2005-01-01

    Traditional lapped transform domain excision techniques obtain good performance at the expense of increased processing delay. Extension of transform domain filtering techniques to the lapped biorthogonal transform domain can help solve the problem. By incorporating biorthogonality into the lapped transforms, more flexibility is obtained in the design of windows. Thus transform bases with better stopband attenuation can be generated by designing windows, but not by increasing the overlapping factor. In this paper, a new modulated lapped biorthogonal transform (MLBT) with optimized windows is introduced for efficient compression of multi-tone interfering signal energy. The bit error rate (BER) performance of the receiver employing the proposed MLBT excision technique is analyzed and compared with that of the lapped transform domain excision-based receivers. Simulation results demonstrate the improved performance and increased robustness of the proposed technique.

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

    Science.gov (United States)

    Swinger, Kerren K; Rice, Phoebe A

    2007-01-26

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

  10. Structure-based Analysis to Hu-DNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Swinger,K.; Rice, P.

    2007-01-01

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

  11. DNA-Based Photonic Bandgap Structures and Devices

    Science.gov (United States)

    2009-11-29

    Genes to Machines: DNA Nanomechanical Devices, Trends in Biochemical Sciences 30, 119-125 (2005). 4. N.C. Seeman. Structural DNA Nanotechnology: An... kpc ≥ ω , k becomes purely real.. If the dispersion relation just given is written as =++ 22)( kpkak 1ε 2)( c ω , it resembles that for modes in a...waveguide. By analogy, the frequency region for which 1ε 22)( kpc < ω will be referred to as cutoff. IV. APPLICATIONS The presence of molecules

  12. A Novel DNA-Based Vaccine Methodology for Aids

    Science.gov (United States)

    1998-11-01

    delivery of DNA to the tongue suggests the tongue may be an inductive site for mucosal immunity . 18 To evaluate the immune effects of IL-6, we delivered DNA...indicate the enhanced protection seen with IL-6 and tongue delivery is not due to induction of localized mucosal immunity . These results further suggest...that the tongue is not an inductive site for mucosal immunity . However, these results do not account for potential differences between the mouse and

  13. Nano-Bio Electronic Devices Based on DNA Bases and Proteins

    Science.gov (United States)

    Rinaldi, R.; Maruccio, G.; Bramanti, A.; Visconti, P.; Biasco, A.; Arima, V.; D'Amico, S.; Cingolani, R.

    A key challenge of the current research in nanoelectronics is the realization of biomolecular devices. The biomolecules have specific functionalies that can be exploited for the implementation of electronic and optoelectronic devices. Different nanotechnological strategies have been pursued to implement the biomolecular devices, following a bottom-up or a topdown approach depending on the used biomolecule and on its functionality. In this paper we present our results on the implementation of nano-biomolecular devices based on modified DNA nucleosides and metalloproteins.

  14. Sequence-specific DNA interactions with calixarene-based langmuir monolayers.

    Science.gov (United States)

    Rullaud, Vanessa; Moridi, Negar; Shahgaldian, Patrick

    2014-07-29

    The interactions of an amphiphilic calixarene, namely p-guanidino-dodecyloxy-calix[4]arene, 1, self-assembled as Langmuir monolayers, with short double stranded DNA, were investigated by surface pressure-area (π-A) isotherms, surface ellipsometry and Brewster angle microscopy (BAM). Three DNA 30mers were used as models, poly(AT), poly(GC) and a random DNA sequence with 50% of G:C base pairs. The interactions of these model DNA duplexes with 1-based Langmuir monolayers were studied by measuring compression isotherms using increasing DNA concentrations (10(-6), 10(-5), 10(-4), and 5 × 10(-4) g L(-1)) in the aqueous subphase. The isotherms of 1 showed an expansion of the monolayer with, interestingly, significant differences depending on the duplex DNA sequence studied. Indeed, the interactions of 1-based monolayers with poly(AT) led to an expansion of the monolayer that was significantly more pronounced that for monolayers on subphases of poly(GC) and the random DNA sequence. The structure and thickness of 1-based Langmuir monolayers were investigated by BAM and surface ellipsometry that showed differences in thickness and structure between a monolayer formed on pure water or on a DNA subphase, with here again relevant dissimilarities depending on the DNA composition.

  15. DNA origami-based standards for quantitative fluorescence microscopy.

    Science.gov (United States)

    Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

    2014-01-01

    Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

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

    Energy Technology Data Exchange (ETDEWEB)

    1987-01-01

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

  17. Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

    Science.gov (United States)

    Skotadis, Evangelos; Voutyras, Konstantinos; Chatzipetrou, Marianneza; Tsekenis, Georgios; Patsiouras, Lampros; Madianos, Leonidas; Chatzandroulis, Stavros; Zergioti, Ioanna; Tsoukalas, Dimitris

    2016-07-15

    A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA.

  18. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

    Science.gov (United States)

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

    2016-12-15

    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  19. Linearly programmed DNA-based molecular computer operated on magnetic particle surface in test-tube

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jian; ZHANG Zhizhou; SHI Yongyong; Li Xiuxia; HE Lin

    2004-01-01

    The postgenomic era has seen an emergence of new applications of DNA manipulation technologies, including DNA-based molecular computing. Surface DNA computing has already been reported in a number of studies that, however, all employ different mechanisms other than automaton functions. Here we describe a programmable DNA surface-computing device as a Turing machine-like finite automaton. The laboratory automaton is primarily composed of DNA (inputs, output-detectors, transition molecules as software), DNA manipulating enzymes and buffer system that solve artificial computational problems autonomously. When fluoresceins were labeled in the 5′ end of (-) strand of the input molecule, direct observation of all reaction intermediates along the time scale was made so that the dynamic process of DNA computing could be conveniently visualized. The features of this study are: (i) achievement of finite automaton functions by linearly programmed DNA computer operated on magnetic particle surface and (ii) direct detection of all DNA computing intermediates by capillary electrophoresis. Since DNA computing has the massive parallelism and feasibility for automation, this achievement sets a basis for large-scale implications of DNA computing for functional genomics in the near future.

  20. Endoscopic-assisted excision of esthesioneuroblastoma.

    Science.gov (United States)

    Prasad, Kishore Chandra; Kumar, Ashwini; Prasad, Sampath Chandra; Jain, Disha

    2007-09-01

    The purpose of this article is to report a case of esthesioneuroblastoma involving the bilateral paranasal sinuses, which was excised using an endoscopic-assisted transfacial approach. A patient presented with nasal swelling and left-sided nasal obstruction, epistaxis, and diplopia. Examination revealed broadening of the nasal dorsum with a fleshy pink mass in both nasal cavities. Computed tomographic scan showed a mass involving the nasal cavity and paranasal sinuses on both sides. The tumor was diagnosed as group C esthesioneuroblastoma. The mass was excised by bilateral medial maxillectomy and bilateral frontoethmoidectomy. Using a 0 degrees endoscope, the attachment of the tumor to the cribriform plate was identified and resected using a motordrill. On Waroff staining, Hispathology slides suggested esthesioneuroblastoma. The patient was asymptomatic for 1 year, following which he developed infection of the nasal cavity for which he had no form of treatment. He subsequently developed maggots in the nasal cavity after which he died. An endoscopic resection of the cribriform plate from the nasal cavity without a formal craniofacial resection can be safely performed with oncologic safety.

  1. Synthesis, photochemical properties and DNA binding studies of dna cleaving agents based on chiral dipyridine dihydrodioxins salts

    Science.gov (United States)

    Shamaev, Alexei

    activated by UV-light. The mechanism of o-quinone release and intramolecular ET was studied in detail by methods of Ultrafast Transient Absortion Spectroscopy and supported by high-level quantum mechanical calculations. The binding properties of chiral intercalators based on PDHD to various DNA oligonucleotides were studied by various methods and DNA cleavage properties indicating strong binding and cleaving ability of the synthesized PDHDs. Also, a new method for synthesis of cyclohexa[e]pyrenes which possibly capable of intramolecular ET and electron transfer-oxidative stress (ET-OS) DNA cleavage was developed and partially accomplished.

  2. An Experimental Population Study of Nucleotide Excision Repair as a Risk Factor for UVB-induced Melanoma

    OpenAIRE

    Fernandez, André A.; Garcia, Rachel; Paniker, Lakshmi; Trono, David; Mitchell, David L.

    2011-01-01

    Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in-situ and in-vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB inducible melanoma mod...

  3. CUPRAC colorimetric and electroanalytical methods determining antioxidant activity based on prevention of oxidative DNA damage.

    Science.gov (United States)

    Uzunboy, Seda; Çekiç, Sema Demirci; Eksin, Ece; Erdem, Arzum; Apak, Reşat

    2017-02-01

    An unbalanced excess of oxygen/nitrogen species (ROS/RNS) can give oxidative hazard to DNA and other biomacromolecules under oxidative stress conditions. While the 'comet' assay for measuring DNA damage is neither specific nor practical, monitoring oxidative changes on individual DNA bases and other oxidation products needs highly specialized equipment and operators. Thus, we developed a modified CUPRAC (cupric ion reducing antioxidant capacity) colorimetric method to determine the average total damage on DNA produced by Fenton oxidation, taking advantage of the fact that the degradation products of DNA but not the original macromolecule is CUPRAC-responsive. The DNA-protective effects of water-soluble antioxidants were used to devise a novel antioxidant activity assay, considered to be physiologically more realistic than those using artificial probes. Our method, based on the measurement of DNA oxidative products with CUPRAC colorimetry proved to be 2 orders-of-magnitude more sensitive than the widely used TBARS (thiobarbituric acid-reactive substances) colorimetric assay used as reference. Additionally, the DNA damage was electrochemically investigated using pencil graphite electrodes (PGEs) as DNA sensor platform in combination with differential pulse voltammetry (DPV). The interaction of the radical species with DNA in the absence/presence of antioxidants was detected according to the changes in guanine oxidation signal.

  4. Predicting DNA-binding sites of proteins based on sequential and 3D structural information.

    Science.gov (United States)

    Li, Bi-Qing; Feng, Kai-Yan; Ding, Juan; Cai, Yu-Dong

    2014-06-01

    Protein-DNA interactions play important roles in many biological processes. To understand the molecular mechanisms of protein-DNA interaction, it is necessary to identify the DNA-binding sites in DNA-binding proteins. In the last decade, computational approaches have been developed to predict protein-DNA-binding sites based solely on protein sequences. In this study, we developed a novel predictor based on support vector machine algorithm coupled with the maximum relevance minimum redundancy method followed by incremental feature selection. We incorporated not only features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, solvent accessibility, but also five three-dimensional (3D) structural features calculated from PDB data to predict the protein-DNA interaction sites. Feature analysis showed that 3D structural features indeed contributed to the prediction of DNA-binding site and it was demonstrated that the prediction performance was better with 3D structural features than without them. It was also shown via analysis of features from each site that the features of DNA-binding site itself contribute the most to the prediction. Our prediction method may become a useful tool for identifying the DNA-binding sites and the feature analysis described in this paper may provide useful insights for in-depth investigations into the mechanisms of protein-DNA interaction.

  5. DNA quantification based on FRET realized by combination with surfactant CPB.

    Science.gov (United States)

    Liu, Chunxia; Wang, Lei; Jiang, Wei

    2010-04-15

    In this work, we developed a novel DNA quantitative analysis based on fluorescence resonance energy transfer (FRET) realized by combination with a surfactant CPB. The approach was capable of detecting long-stranded DNA in a separation-free format. A sandwich-type FAM-c-DNA-t-DNA-r-DNA-TAMRA conjugate was first formed by the capture probe tagged with FAM, the reporter probe tagged with TAMRA and the target DNA through hybridization. The donor (FAM) and the acceptor (TAMRA) were bridged to afford a FRET system. Subsequently, an addition of the cationic surfactant CPB to the system resulted in a substantial change of the microenvironment and an effective condensation of DNA strands. Consequently, without altering the component of the double strands, an enhanced acceptor fluorescence signal from FRET was achieved and a quantification of the target DNA containing 30 bases was enabled. Under the optimal experimental conditions, an excellent linear relationship between the increase of acceptor fluorescent peak area and the target DNA concentration was obtained over the range from 1.0 x 10(-7) to 3.0 x 10(-9) mol L(-1). The proposed approach offered adequate sensitivity for the detection of the target DNA at 1.0 x 10(-9) mol L(-1).

  6. Luminescent Iridium(III) Complex Labeled DNA for Graphene Oxide-Based Biosensors.

    Science.gov (United States)

    Zhao, Qingcheng; Zhou, Yuyang; Li, Yingying; Gu, Wei; Zhang, Qi; Liu, Jian

    2016-02-02

    There has been growing interest in utilizing highly photostable iridium(III) complexes as new luminescent probes for biotechnology and life science. Herein, iridium(III) complex with carboxyl group was synthesized and activated with N-hydroxysuccinimide, followed by tagging to the amino terminate of single-stranded DNA (ssDNA). The Ir-ssDNA probe was further combined with graphene oxide (GO) nanosheets to develop a GO-based biosensor for target ssDNA detection. The quenching efficiency of GO, and the photostability of iridium(III) complex and GO-Ir-ssDNA biosensor, were also investigated. On the basis of the high luminescence quenching efficiency of GO toward iridium(III) complex, the GO-Ir-ssDNA biosensor exhibited minimal background signals, while strong emission was observed when Ir-ssDNA desorbed from GO nanosheets and formed a double helix with the specific target, leading to a high signal-to-background ratio. Moreover, it was found that luminescent intensities of iridium(III) complex and GO-Ir-ssDNA biosensor were around 15 and 3 times higher than those of the traditional carboxyl fluorescein (FAM) dye and the GO-FAM-ssDNA biosensor after UV irradiation, respectively. Our study suggested the sensitive and selective Ir-ssDNA probe was suitable for the development of highly photostable GO-based detection platforms, showing promise for application beyond the OLED (organic light emitting diode) area.

  7. Transgenerational inheritance: Models and mechanisms of non-DNA sequence-based inheritance.

    Science.gov (United States)

    Miska, Eric A; Ferguson-Smith, Anne C

    2016-10-07

    Heritability has traditionally been thought to be a characteristic feature of the genetic material of an organism-notably, its DNA. However, it is now clear that inheritance not based on DNA sequence exists in multiple organisms, with examples found in microbes, plants, and invertebrate and vertebrate animals. In mammals, the molecular mechanisms have been challenging to elucidate, in part due to difficulties in designing robust models and approaches. Here we review some of the evidence, concepts, and potential mechanisms of non-DNA sequence-based transgenerational inheritance. We highlight model systems and discuss whether phenotypes are replicated or reconstructed over successive generations, as well as whether mechanisms operate at transcriptional and/or posttranscriptional levels. Finally, we explore the short- and long-term implications of non-DNA sequence-based inheritance. Understanding the effects of non-DNA sequence-based mechanisms is key to a full appreciation of heritability in health and disease.

  8. Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)

    OpenAIRE

    Enfors Sven-Olof; Wegrzyn Grzegorz; Basselet Pascal; Gabig-Ciminska Magdalena

    2008-01-01

    Abstract Background Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. Results A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated co...

  9. Staged excisions of moderate-sized burns compared with total excision with immediate autograft: an evaluation of two strategies

    Science.gov (United States)

    Elmasry, Moustafa; Steinvall, Ingrid; Thorfinn, Johan; Abdelrahman, Islam; Olofsson, Pia; Sjoberg, Folke

    2017-01-01

    Background: Different surgical techniques have evolved since excision and autografting became the treatment of choice for deep burns in the 1970s. The treatment plan at the Burn Center, Linköping University Hospital, Sweden, has shifted from single-stage excision and immediate autografting to staged excisions and temporary cover with xenografts before autografting. The aim of this study was to find out if the change in policy resulted in extended duration of hospital stay/total body surface area burned (LOS/TBSA%). Methods: Retrospective clinical cohort including surgically-managed patients with burns of 15%-60% TBSA% within each treatment group. The first had early full excisions of deep dermal and full thickness burns and immediate autografts (1997-98), excision and immediate autograft group) and the second had staged excisions before final autografts using xenografts for temporary cover (2010-11, staged excision group). Results: The study included 57 patients with deep dermal and full-thickness burns, 28 of whom had excision and immediate autografting, and 29 of whom had staged excisions with xenografting before final autografting. Adjusted (LOS/TBSA%) was close to 1, and did not differ between groups. Mean operating time for the staged excision group was shorter and the excised area/operation was smaller. The total operating time/TBSA% did not differ between groups. Conclusion: Staged excisions with temporary cover did not affect adjusted LOS/TBSA% or total operating time. Staged excisions may be thought to be more expensive because of the cost of covering the wound between stages, but this needs to be further investigated as do the factors that predict long term outcome. PMID:28123862

  10. Precise excision of transposons and point mutations induced by chemicals.

    Science.gov (United States)

    Rusina OYu; Mirskaya, E E; Andreeva, I V; Skavronskaya, A G

    1992-11-01

    The ability of 23 chemicals (carcinogens and non-carcinogens) to induce precise excision of Tn10 and point mutations was studied in experiments with a single strain. The mutation assay was shown to detect a wider spectrum of genotoxic agents than the assay of Tn10 precise excision. The latter was induced only by potent SOS mutagens, which is in accordance with data on the SOS dependence of the induction of precise excision of Tn10. The precise excision assay as an additional test contributing to the knowledge of particular features of the action of a tested mutagen is discussed. The induction of precise excision of Tn10 by pyrene (and its failure to induce point mutations in this strain) demonstrates the value of using the transposon excision assay in cases of 'problem' mutagens.

  11. Multispectral and Photoplethysmography Optical Imaging Techniques Identify Important Tissue Characteristics in an Animal Model of Tangential Burn Excision.

    Science.gov (United States)

    Thatcher, Jeffrey E; Li, Weizhi; Rodriguez-Vaqueiro, Yolanda; Squiers, John J; Mo, Weirong; Lu, Yang; Plant, Kevin D; Sellke, Eric; King, Darlene R; Fan, Wensheng; Martinez-Lorenzo, Jose A; DiMaio, J Michael

    2016-01-01

    Burn excision, a difficult technique owing to the training required to identify the extent and depth of injury, will benefit from a tool that can cue the surgeon as to where and how much to resect. We explored two rapid and noninvasive optical imaging techniques in their ability to identify burn tissue from the viable wound bed using an animal model of tangential burn excision. Photoplethysmography (PPG) imaging and multispectral imaging (MSI) were used to image the initial, intermediate, and final stages of burn excision of a deep partial-thickness burn. PPG imaging maps blood flow in the skin's microcirculation, and MSI collects the tissue reflectance spectrum in visible and infrared wavelengths of light to classify tissue based on a reference library. A porcine deep partial-thickness burn model was generated and serial tangential excision accomplished with an electric dermatome set to 1.0 mm depth. Excised eschar was stained with hematoxylin and eosin to determine the extent of burn remaining at each excision depth. We confirmed that the PPG imaging device showed significantly less blood flow where burn tissue was present, and the MSI method could delineate burn tissue in the wound bed from the viable wound bed. These results were confirmed independently by a histological analysis. We found these devices can identify the proper depth of excision, and their images could cue a surgeon as to the preparedness of the wound bed for grafting. These image outputs are expected to facilitate clinical judgment in the operating room.

  12. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents.

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; Reis, Ana Beatriz de Bragança Dos; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-05-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

  13. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  14. Surgical Excision of Multiple Penile Syringomas With Scrotal Flap Reconstruction

    OpenAIRE

    2014-01-01

    Objective: Penile syringomas are rare lesions usually occurring in isolation. We report the excision and reconstruction of multiple synchronous penile shaft syringomas with local scrotal flaps. Methods: We report a rare case of excision of multiple penile syringomas and reconstruction with scrotal flaps in a 29-year-old man. Results: Penile syringomas were excised and reconstructed with scrotal flaps in a single-stage procedure. Conclusions: In addition to providing wound coverage, this recon...

  15. Protein sequence for clustering DNA based on Artificial Neural Networks

    Directory of Open Access Journals (Sweden)

    Gamal. F. Elhadi

    2012-01-01

    Full Text Available DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. Clustering is a process that groups a set of objects into clusters so that the similarity among objects in the same cluster is high, while that among the objects in different clusters is low. In this paper, we proposed an approach for clustering DNA sequences using Self-Organizing Map (SOM algorithm and Protein Sequence. The main objective is to analyze biological data and to bunch DNA to many clusters more easily and efficiently. We use the proposed approach to analyze both large and small amount of input DNA sequences. The results show that the similarity of the sequences does not depend on the amount of input sequences. Our approach depends on evaluating the degree of the DNA sequences similarity using the hierarchal representation Dendrogram. Representing large amount of data using hierarchal tree gives the ability to compare large sequences efficiently

  16. Advancing DNA-based Nanotechnology Capabilities and Applications

    Science.gov (United States)

    Marchi, Alexandria N.

    Biological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.

  17. Accurate Evolutions of Orbiting Black-Hole Binaries Without Excision

    CERN Document Server

    Campanelli, M; Marronetti, P; Zlochower, Y

    2006-01-01

    We present a new algorithm for evolving orbiting black-hole binaries that does not require excision or a corotating shift. Our algorithm is based on a novel technique to handle the singular puncture conformal factor. This system, based on the BSSN formulation of Einstein's equations, when used with a `pre-collapsed' initial lapse, is non-singular at the start of the evolution, and remains non-singular and stable provided that a good choice is made for the gauge. As a test case, we use this technique to fully evolve orbiting black-hole binaries from near the Innermost Stable Circular Orbit (ISCO) regime. We show fourth order convergence of waveforms and compute the radiated gravitational energy and angular momentum from the plunge. These results are in good agreement with those predicted by the Lazarus approach.

  18. Polymorphisms in nucleotide excision repair genes, smoking and intake of fruit and vegetables in relation to lung cancer

    DEFF Research Database (Denmark)

    Raaschou-Nielsen, Ole; Sørensen, Mette; Overvad, Kim;

    2007-01-01

    Polymorphisms in nucleotide excision repair genes have been associated with risk for lung cancer. We examined gene-environment interactions in relation to lung cancer in 430 cases and 790 comparison persons identified within a prospective cohort of 57,053 persons. We included polymorphisms...... in the XPC, XPA and XPD genes involved in the nucleotide excision DNA repair pathway and analysed possible interactions with smoking and dietary intake of fruit and vegetables in relation to risk for lung cancer. We found that intake of fruit was associated with lower risk for lung cancer only among carriers...

  19. DNA origami-based nanoribbons: assembly, length distribution, and twist

    Energy Technology Data Exchange (ETDEWEB)

    Jungmann, Ralf; Scheible, Max; Kuzyk, Anton; Pardatscher, Guenther; Simmel, Friedrich C [Lehrstuhl fuer Bioelektronik, Physik-Department and ZNN/WSI, Technische Universitaet Muenchen, Am Coulombwall 4a, 85748 Garching (Germany); Castro, Carlos E, E-mail: simmel@ph.tum.de [Labor fuer Biomolekulare Nanotechnologie, Physik-Department and ZNN/WSI, Technische Universitaet Muenchen, Am Coulombwall 4a, 85748 Garching (Germany)

    2011-07-08

    A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.

  20. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  1. Binding of copper(II) polypyridyl complexes to DNA and consequences for DNA-based asymmetric catalysis.

    Science.gov (United States)

    Draksharapu, Apparao; Boersma, Arnold J; Leising, Miriam; Meetsma, Auke; Browne, Wesley R; Roelfes, Gerard

    2015-02-28

    The interaction between salmon testes DNA (st-DNA) and a series of Cu(II) polypyridyl complexes, i.e. [Cu(dmbpy)(NO3)2] (1) (dmbpy = 4,4'-dimethyl-2,2'-bipyridine), [Cu(bpy)(NO3)2] (2) (bpy = 2,2'-bipyridine), [Cu(phen)(NO3)2] (3) (phen = phenanthroline), [Cu(terpy)(NO3)2]·H2O (4) (terpy = 2,2':6',2″-terpyridine), [Cu(dpq)(NO3)2] (5) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) and [Cu(dppz)(NO3)2] (6) (dppz = dipyrido[3,2-a:2',3'-c]phenazine) was studied by UV/Vis absorption, Circular Dichroism, Linear Dichroism, EPR, Raman and (UV and vis) resonance Raman spectroscopies and viscometry. These complexes catalyse enantioselective C-C bond forming reactions in water with DNA as the source of chirality. Complex 1 crystallizes as an inorganic polymer with nitrate ligands bridging the copper ions, which adopt essentially a distorted square pyramidal structure with a fifth bridging nitrate ligand at the axial position. Raman spectroscopy indicates that in solution the nitrate ligands in 1, 2, 3 and 4 are displaced by solvent (H2O). For complex 1, multiple supramolecular species are observed in the presence of st-DNA in contrast to the other complexes, which appear to interact relatively uniformly as a single species predominantly, when st-DNA is present. Overall the data suggest that complexes 1 and 2 engage primarily through groove binding with st-DNA while 5 and 6 undergo intercalation. For complexes 3 and 4 the data indicates that both groove binding and intercalation takes place, albeit primarily intercalation. Although it is tempting to conclude that the groove binders give highest ee and rate acceleration, it is proposed that the flexibility and dynamics in binding of Cu(II) complexes to DNA are key parameters that determine the outcome of the reaction. These findings provide insight into the complex supramolecular structure of these DNA-based catalysts.

  2. A surface-based DNA algorithm for the minimal vertex cover problem

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    DNA computing was proposed for solving a class of intractable computational problems, of which the computing time will grow exponentially with the problem size. Up to now, many achievements have been made to improve its performance and increase its reliability. It has been shown many times that the surface-based DNA computing technique has very low error rate, but the technique has not been widely used in the DNA computing algorithms design. In this paper, a surface-based DNA computing algorithm for minimal vertex cover problem, a problem well-known for its exponential difficulty, is introduced. This work provides further evidence for the ability of surface-based DNA computing in solving NP-complete problems.

  3. Exponential quadruplex priming amplification for DNA-based isothermal diagnostics.

    Science.gov (United States)

    Partskhaladze, Tamar; Taylor, Adam; Lomidze, Levan; Gvarjaladze, David; Kankia, Besik

    2015-02-01

    Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point-of-care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self-dissociate from the primer-binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 10(10)-fold amplification of DNA signal in less than 40 min.

  4. Single-strand DNA detection using a planar photonic-crystal-waveguide-based sensor.

    Science.gov (United States)

    Toccafondo, V; García-Rupérez, J; Bañuls, M J; Griol, A; Castelló, J G; Peransi-Llopis, S; Maquieira, A

    2010-11-01

    We report an experimental demonstration of single-strand DNA (ssDNA) detection at room temperature using a photonic-crystal-waveguide-based optical sensor. The sensor surface was previously biofunctionalized with ssDNA probes to be used as specific target receptors. Our experiments showed that it is possible to detect these hybridization events using planar photonic-crystal structures, reaching an estimated detection limit as low as 19.8 nM for the detection of the complementary DNA strand.

  5. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    Science.gov (United States)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  6. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

    Science.gov (United States)

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga

    2011-06-01

    DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

  7. Quantum dot based DNA nanosensors for amplification-free detection of human topoisomerase I

    DEFF Research Database (Denmark)

    Jepsen, Morten Leth; Ottaviani, Alessio; Knudsen, Birgitta R.;

    2014-01-01

    We develop a quantum dot based DNA nanosensor specifically targeting the cleavage–religation activity of an essential DNA-modifying enzyme, human topoisomerase I. The assay has shown great promise in biological crude samples and thus is expected to contribute to clinical diagnostics and anti...

  8. Investigation of the charge effect on the electrochemical transduction in a quinone-based DNA sensor

    DEFF Research Database (Denmark)

    Reisberg, S.; Piro, B.; Noel, V.;

    2008-01-01

    To elucidate the mechanism involved in the electrochemical transduction process of a conducting polymer-based DNA sensor, peptide nucleic acids (PNA) were used. PNA are DNA analogues having similar hybridization properties but are neutral. This allows to discriminate the electrostatic effect of D...

  9. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

    NARCIS (Netherlands)

    Yu, S.C.; Chan, K.C.; Zheng, Y.W.; Jiang, P.; Liao, G.J.; Sun, H; Akolekar, R.; Leung, T.Y.; Go, A.T.; Vugt, J.M.G. van; Minekawa, R.; Oudejans, C.B.; Nicolaides, K.H.; Chiu, R.W.; Lo, Y.M.

    2014-01-01

    Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach th

  10. Base-resolution DNA methylation landscape of zebrafish brain and liver

    Directory of Open Access Journals (Sweden)

    Aniruddha Chatterjee

    2014-12-01

    To our knowledge, these datasets are the only RRBS datasets and base-resolution DNA methylation data available at this time for zebrafish brain and liver. These datasets could serve as a resource for future studies to document the functional role of DNA methylation in zebrafish. In addition, these datasets could be used as controls while performing analysis on treated samples.

  11. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

    Science.gov (United States)

    Yu, Stephanie C Y; Chan, K C Allen; Zheng, Yama W L; Jiang, Peiyong; Liao, Gary J W; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y; Go, Attie T J I; van Vugt, John M G; Minekawa, Ryoko; Oudejans, Cees B M; Nicolaides, Kypros H; Chiu, Rossa W K; Lo, Y M Dennis

    2014-06-10

    Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

  12. A New Revised DNA Cramp Tool Based Approach of Chopping DNA Repetitive and Non-Repetitive Genome Sequences

    Directory of Open Access Journals (Sweden)

    V.Hari Prasad

    2012-11-01

    Full Text Available In vogue tremendous amount of data generated day by day by the living organism of genetic sequences and its accumulation in database, their size is growing in an exponential manner. Due to excessive storage of DNA sequences in public databases like NCBI, EMBL and DDBJ archival maintenance is tedious task. Transmission of information from one place to another place in network management systems is also a critical task. So To improve the efficiency and to reduce the overhead of the database need of compression arises in database optimization. In this connection different techniques were bloomed, but achieved results are not bountiful. Many classical algorithms are fails to compress genetic sequences due to the specificity of text encoded in dna and few of the existing techniques achieved positive results. DNA is repetitive and non repetitive in nature. Our proposed technique DNACRAMP is applicable on repetitive and non repetitive sequences of dna and it yields better compression ratio in terms of bits per bases. This is compared with existing techniques and observed that our one is the optimum technique and compression results are on par with existing techniques.

  13. Adsorption of DNA on colloidal Ag nanoparticles: effects of nanoparticle surface charge, base content and length of DNA.

    Science.gov (United States)

    Abbasian, Sara; Moshaii, Ahmad; Nikkhah, Maryam; Farkhari, Nahid

    2014-04-01

    The adsorption of single and double stranded DNA on colloidal silver nanoparticles has been studied to investigate the effects of surface charge of the nanoparticles, the composition of the oligonucleotide and its length on the adsorption characteristics. The results explain that the nanoparticle surface charge is a key parameter determining the propensity of oligonucleotides to adsorb on nanoparticles. The adsorption also depends on the length and composition of oligonucleotide. The protective effects of both single and double stranded DNA against salt-induced aggregation dramatically increase as the DNA length increases. In contrast to other available reports, we observed that long oligonucleotides (single-stranded and double stranded) can well be adsorbed on the nanoparticles as the short ones leading to almost complete protection of nanoparticles against salt induced aggregation and hence are not suitable for the sensing applications. Finally, the light scattering from the Ag nanoparticles has been simulated and the results compared with the experiments. Our understanding should improve development of colorimetric assays for DNA detection based on aggregation of unmodified metallic nanoparticles.

  14. Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

    DEFF Research Database (Denmark)

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina

    2013-01-01

    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient...... in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex® 100 matrix, Phenol......-Cloroform-Isoamyl alcohol, NucliSENS® magnetic extraction kit, and DNeasy® Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy® Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However...

  15. Feasibility of using DNA-immobilized nanocellulose-based immunoadsorbent for systemic lupus erythematosus plasmapheresis.

    Science.gov (United States)

    Xu, Changgang; Carlsson, Daniel O; Mihranyan, Albert

    2016-07-01

    The goal of this project was to study the feasibility of using a DNA-immobilized nanocellulose-based immunoadsorbent for possible application in medical apheresis such as systemic lupus erythematosus (SLE) treatment. Calf thymus DNA was bound to high surface area nanocellulose membrane at varying concentrations using UV-irradiation. The DNA-immobilized samples were characterized with scanning electron microscopy, atomic force microscopy, and phosphorus elemental analysis. The anti-ds-DNA IgG binding was tested in vitro using ELISA. The produced sample showed high affinity in vitro to bind anti-ds-DNA-antibodies from mice, as much as 80% of added IgG was bound by the membrane. Furthermore, the binding efficiency was quantitatively dependent on the amount of immobilized DNA onto nanocellulose membrane. The described nanocellulose membranes are interesting immunoadsorbents for continued clinical studies.

  16. [Under what conditions does G.C Watson-Crick DNA base pair acquire all four configurations characteristic for A.T Watson-Crick DNA base pair?].

    Science.gov (United States)

    Brovarets', O O

    2013-01-01

    At the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory it was established for the first time, that the Löwdin's G*.C* DNA base pair formed by the mutagenic tautomers can acquire, as the A-T Watson-Crick DNA base pair, four biologically important configurations, namely: Watson-Crick, reverse Watson-Crick, Hoogsteen and reverse Hoogsteen. This fact demonstrates rather unexpected role of the tautomerisation of the one of the Watson-Crick DNA base pairs, in particular, via double proton transfer: exactly the G.C-->G*.C* tautomerisation allows to overcome steric hindrances for the implementation of the above mentioned configurations. Geometric, electron-topological and energetic properties of the H-bonds that stabilise the studied pairs, as well as the energetic characteristics of the latters are presented.

  17. A chiroptical switch based on DNA/layered double hydroxide ultrathin films.

    Science.gov (United States)

    Shi, Wenying; Jia, Yankun; Xu, Simin; Li, Zhixiong; Fu, Yi; Wei, Min; Shi, Shuxian

    2014-11-04

    A highly oriented film was fabricated by layer-by-layer self-assembly of DNA and MgAl-layered double hydroxide nanosheets, and its application in chiroptical switch was demonstrated via intercalation and deintercalation of an achiral molecule into the DNA cavity. DNA molecules are prone to forming an ordered and dispersive state in the interlayer region of rigid layered double hydroxide (LDH) nanosheets as confirmed by scanning electron microscopy and atomic force microscopy. The induced chiroptical ultrathin film (UTF) is achieved via the intercalation of an achiral chromophore [5,10,15,20-tetrakis(4-N-methylpyridyl)porphine tetra(p-toluenesulfonate) (TMPyP)] into the spiral cavity of DNA stabilized in the LDH matrix [denoted as TMPyP-(DNA/LDH)20]. Fluorescence and circular dichroism spectroscopy are utilized to testify the intercalation of TMPyP into (DNA/LDH)20 UTF that involves two steps: the electrostatic binding of TMPyP onto the surface of (DNA/LDH)20 followed by intercalation into base pairs of DNA. In addition, the TMPyP-(DNA/LDH)20 UTF exhibits good reversibility and repeatability in induced optical chirality, based on the intercalation and deintercalation of TMPyP by alternate exposure to HCl and NH3/H2O vapor, which can be potentially used as a chiroptical switch in data storage.

  18. Conjunctival spheroid degeneration. Recurrence after excision.

    Science.gov (United States)

    Norn, M S

    1982-06-01

    After excision of part of the conjunctiva in 15 eyes (14 subjects) due to spheroid degeneration, the author noticed only fairly small, varying numbers of autofluorescent and colourless spheroids-after an observation period of 18 months, only 6% of autofluorescent and 13% of colourless bodies were observed compared to the number before biopsy. Around the biopsy site only a few spheroids were seen, with a non-significant tendency to increase in number of the colourless bodies. In the cornea the band-shaped keratopathy had aggravated, with the formation of a small number of large, autofluorescent spheroids. A pinguecula recurred in a mild degree only in 3 out of 13 cases within 18 months.

  19. Reversibly locked thionucleobase pairs in DNA to study base flipping enzymes

    Directory of Open Access Journals (Sweden)

    Christine Beuck

    2014-10-01

    Full Text Available Covalently interstrand cross-linked DNA is an interesting tool to study DNA binding proteins that locally open up the DNA duplex by flipping single bases out of the DNA helix or melting whole stretches of base pairs to perform their function. The ideal DNA cross-link to study protein–DNA interactions should be specific and easy to synthesize, be stable during protein binding experiments, have a short covalent linker to avoid steric hindrance of protein binding, and should be available as a mimic for both A/T and G/C base pairs to cover all possible binding specificities. Several covalent interstrand cross-links have been described in the literature, but most of them fall short of at least one of the above criteria. We developed an efficient method to site-specifically and reversibly cross-link thionucleoside base pairs in synthetic duplex oligodeoxynucleotides by bisalkylation with 1,2-diiodoethane resulting in an ethylene-bridged base pair. Both linked A/T and G/C base pair analogs can conveniently be prepared which allows studying any base pair-opening enzyme regardless of its sequence specificity. The cross-link is stable in the absence of reducing agents but the linker can be quickly and tracelessly removed by the addition of thiol reagents like dithiothreitol. This property makes the cross-linking reaction fully reversible and allows for a switching of the linked base pair from locked to unlocked during biochemical experiments. Using the DNA methyltransferase from Thermus aquaticus (M.TaqI as example, we demonstrate that the presented cross-linked DNA with an ethylene-linked A/T base pair analog at the target position is a useful tool to determine the base-flipping equilibrium constant of a base-flipping enzyme which lies mostly on the extrahelical side for M.TaqI.

  20. Practical aspects of DNA-based forensic studies in dentistry

    Science.gov (United States)

    Muruganandhan, J; Sivakumar, G

    2011-01-01

    Forensic dentistry as a science has evolved from simple methods of age estimation and bite-mark analysis, to a new era of genetic and serological investigations. DNA analysis in forensic science requires a sample or source from either an individual (living or dead) or a crime/incident site. The orofacial region is a good source of such material, due to the fact that certain oral tissues are relatively resistant to environmental degradation and destruction by thermal, electrical, and mechanical insult. Dentists may be called upon to provide samples and expert analysis in many such situations. Sources include soft and hard tissues of teeth and jaws, saliva, biopsy material, and mucosal swabs. Tissue samples should be handled with care, and correct protocol in collection and preparation has to be followed. This ensures a high yield of the required DNA. Hard tissues like teeth require specialized procedures to extract the genetic material. Research has shown that there is a wide variation in the quality and quantity of DNA extracted from different individuals from the same site even under similar conditions. This necessitates calibration of the various methods to achieve best results. DNA analysis can provide highly accurate identification if used correctly. Here a description of the various sources in the oral region has been provided from which samples could be forwarded to the forensic laboratory. Most commonly employed techniques of collection and handling for laboratory procedures have been outlined. PMID:22022138

  1. Practical aspects of DNA-based forensic studies in dentistry.

    Science.gov (United States)

    Muruganandhan, J; Sivakumar, G

    2011-01-01

    Forensic dentistry as a science has evolved from simple methods of age estimation and bite-mark analysis, to a new era of genetic and serological investigations. DNA analysis in forensic science requires a sample or source from either an individual (living or dead) or a crime/incident site. The orofacial region is a good source of such material, due to the fact that certain oral tissues are relatively resistant to environmental degradation and destruction by thermal, electrical, and mechanical insult. Dentists may be called upon to provide samples and expert analysis in many such situations. Sources include soft and hard tissues of teeth and jaws, saliva, biopsy material, and mucosal swabs. Tissue samples should be handled with care, and correct protocol in collection and preparation has to be followed. This ensures a high yield of the required DNA. Hard tissues like teeth require specialized procedures to extract the genetic material. Research has shown that there is a wide variation in the quality and quantity of DNA extracted from different individuals from the same site even under similar conditions. This necessitates calibration of the various methods to achieve best results. DNA analysis can provide highly accurate identification if used correctly. Here a description of the various sources in the oral region has been provided from which samples could be forwarded to the forensic laboratory. Most commonly employed techniques of collection and handling for laboratory procedures have been outlined.

  2. [DNA-based diagnosis of hereditary tumour predisposition

    NARCIS (Netherlands)

    Menko, F.H.; Ligtenberg, M.J.L.; Brouwer, T.; Hahn, D.E.; Ausems, M.G.E.M.

    2007-01-01

    Of all forms of cancer, approximately 5% are caused by factors leading to a strong genetic predisposition. DNA diagnosis is currently used in families with hereditary tumour syndromes, such as familial adenomatous polyposis, hereditary non-polyposis colorectal carcinoma (Lynch syndrome), and heredit

  3. An Efficient Approach in Analysis of DNA Base Calling Using Neural Fuzzy Model

    Science.gov (United States)

    2017-01-01

    This paper presented the issues of true representation and a reliable measure for analyzing the DNA base calling is provided. The method implemented dealt with the data set quality in analyzing DNA sequencing, it is investigating solution of the problem of using Neurofuzzy techniques for predicting the confidence value for each base in DNA base calling regarding collecting the data for each base in DNA, and the simulation model of designing the ANFIS contains three subsystems and main system; obtain the three features from the subsystems and in the main system and use the three features to predict the confidence value for each base. This is achieving effective results with high performance in employment. PMID:28261268

  4. Fluorescent detection of copper(II) based on DNA-templated click chemistry and graphene oxide.

    Science.gov (United States)

    Zhou, Lifen; Shen, Qinpeng; Zhao, Peng; Xiang, Bingbing; Nie, Zhou; Huang, Yan; Yao, Shouzhuo

    2013-12-15

    A novel DNA-templated click chemistry strategy for homogenous fluorescent detection of Cu(2+) has been developed based on click ligation-dependent DNA structure switch and the selective quenching ability of graphene oxide (GO) nanosheet. The clickable duplex probe consists of two DNA strands with alkyne and azide group, respectively, and Cu(+)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction can chemically ligate these two strands. Toehold sequence displacement was consequently exploited to achieve DNA structure transformation bearing fluorescent tag FAM. Cu(2+)-induced chemical ligation caused the probe transfer to hybrid structure with single stranded DNA (ssDNA) tail, while only duplex structure was obtained without Cu(2+). This structural difference can be probed by GO-based fluorescence detection due to the preferential binding of GO to ssDNA. Under the optimum conditions, this sensor can sensitively and specifically detect Cu(2+) with a low detection limit of 58 nM and a linear range of 0.1-10 μM. This new strategy is highly sensitive and selective for Cu(2+) detection because of the great specificity of click chemistry and super-quenching ability of GO. Moreover, with the aid of high efficient DNA templated synthesis, the detection process requires only about half an hour which is much quicker than previous click-chemistry-based Cu(2+) sensors.

  5. Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

    Science.gov (United States)

    Faruqi, A F; Datta, H J; Carroll, D; Seidman, M M; Glazer, P M

    2000-02-01

    The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

  6. Population-based study of DNA image cytometry as screening method for esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Lin Zhao; Guo-Qing Wang; Qi Shang; You-Lin Qiao; Wen-Qiang Wei; De-Li Zhao; Chang-Qing Hao; Dong-Mei Lin; Qin-Jing Pan; Xin-Qing Li; Fu-Hua Lei; Jin-Wu Wang

    2012-01-01

    AIM: To explore the DNA image cytometry (DNA-ICM) technique as a primary screening method for esophageal squamous precancerous lesions.METHODS: This study was designed as a populationbased screening study. A total of 582 local residents aged 40 years-69 years were recruited from Linzhou in Henan and Feicheng in Shandong. However, only 452 subjects had results of liquid-based cytology, DNA-ICM and pathology. The sensitivity and specificity of DNAICM were calculated and compared with liquid-based cytology in moderate dysplasia or worse. RESULTS: Sensitivities of DNA-ICM ranging from at least 1 to 4 aneuploid cells were 90.91%, 86.36%, 79.55% and 77.27%, respectively, which were better than that of liquid-based cytology (75%). Specificities of DNA-ICM were 70.83%, 84.07%, 92.65% and 96.81%, but the specificity of liquid-based cytology was 91.91%. The sensitivity and specificity of a combination of liquid-based cytology and DNA-ICM were 84.09% and 85.78%, respectively. CONCLUSION: It is possible to use DNA-ICM technique as a primary screening method for esophageal squamous precancerous lesions.

  7. Optical detection of PNA/DNA hybridization in resonant porous silicon-based devices

    Science.gov (United States)

    Rotiroti, Lucia; Arcari, Paolo; Lamberti, Annalisa; Sanges, Carmen; De Tommasi, Edoardo; Rea, Ilaria; Rendina, Ivo; De Stefano, Luca

    2008-04-01

    The development of label-free optical biosensors could have a great impact on life sciences as well as on screening techniques for medical and environmental applications. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone, resulting in an achiral and uncharged mimic. Due to the uncharged nature of PNA, PNA-DNA duplexes show a better thermal stability respect the DNA-DNA equivalents. In this work, we used an optical biosensor, based on the porous silicon (PSi) nanotechnology, to detect PNA-DNA interactions. PSi optical sensors are based on changes of reflectivity spectrum when they are exposed to the target analytes. The porous silicon surface was chemically modified to covalently link the PNA which acts as a very specific probe for its ligand (cDNA).

  8. An electrochemical DNA biosensor based on gold nanorods decorated graphene oxide sheets for sensing platform.

    Science.gov (United States)

    Han, Xiaowei; Fang, Xian; Shi, Anqi; Wang, Jiao; Zhang, Yuzhong

    2013-12-15

    A simple electrochemical sensor for sensitive and selective DNA detection was constructed based on gold nanorods (Au NRs) decorated graphene oxide (GO) sheets. The high-quality Au NRs-GO nanocomposite was synthesized via the electrostatic self-assembly technique, which is considered a potential sensing platform. Differential pulse voltammetry was used to monitor the DNA hybridization event using methylene blue as an electrochemical indicator. Under optimal conditions, the peak currents of methylene blue were linear with the logarithm of the concentrations of complementary DNA from 1.0 × 10(-9) to 1.0 × 10(-14)M with a detection limit of 3.5 × 10(-15)M (signal/noise=3). Moreover, the prepared electrochemical sensor can effectively distinguish complementary DNA sequences in the presence of a large amount of single-base mismatched DNA (1000:1), indicating that the biosensor has high selectivity.

  9. Excising a boosted rotating black hole with overlapping grids

    CERN Document Server

    Calabrese, G; Calabrese, Gioel; Neilsen, David

    2004-01-01

    We use the overlapping grids method to construct a fourth order accurate discretization of a first order reduction of the Klein-Gordon scalar field equation on a boosted spinning black hole blackground in axisymmetry. This method allows us to use a spherical outer boundary and excise the singularity from the domain with a spheroidal inner boundary which is moving with respect to the main grid. We discuss the use of higher order accurate energy conserving schemes to handle the axis of symmetry and compare it with a simpler technique based on regularity conditions. We also compare the single grid long term stability property of this formulation of the wave equation with that of a different first order reduction.

  10. Terahertz pulsed imaging of freshly excised human colonic tissues

    Energy Technology Data Exchange (ETDEWEB)

    Reid, Caroline B; Gibson, Adam P [Department of Medical Physics and Bioengineering, University College London, London, WC1E 6BT (United Kingdom); Fitzgerald, Anthony; Wallace, Vincent P [School of Physics, University of Western Australia, Crawley 6009 (Australia); Reese, George; Tekkis, Paris [Division of Surgery, Chelsea and Westminster Campus, Imperial College London, London (United Kingdom); Goldin, Robert [Centre for Pathology, Imperial College London, St Mary' s Campus, London (United Kingdom); O' Kelly, P S [TeraView Ltd, Platinum Building, St John' s Innovation Park, Cowley Road, Cambridge, CB4 0WS (United Kingdom); Pickwell-MacPherson, Emma, E-mail: c.reid@medphys.ucl.ac.uk [Department of Electronic Engineering, Chinese University of Hong Kong, Shatin, NT (Hong Kong)

    2011-07-21

    We present the results from a feasibility study which measures properties in the terahertz frequency range of excised cancerous, dysplastic and healthy colonic tissues from 30 patients. We compare their absorption and refractive index spectra to identify trends which may enable different tissue types to be distinguished. In addition, we present statistical models based on variations between up to 17 parameters calculated from the reflected time and frequency domain signals of all the measured tissues. These models produce a sensitivity of 82% and a specificity of 77% in distinguishing between healthy and all diseased tissues and a sensitivity of 89% and a specificity of 71% in distinguishing between dysplastic and healthy tissues. The contrast between the tissue types was supported by histological staining studies which showed an increased vascularity in regions of increased terahertz absorption.

  11. Effect of structure on sensing performance of a target induced signaling probe shifting DNA-based (TISPS-DNA) sensor.

    Science.gov (United States)

    Yu, Xiang; Yu, Zhigang; Li, Fengqin; Xu, Yanmei; He, Xunjun; Xu, Lan; Shi, Wenbing; Zhang, Guiling; Yan, Hong

    2017-05-15

    A type of "signal on" displacement-based sensors named target induced signaling probe shifting DNA-based (TISPS-DNA) sensor were developed for a designated DNA detection. The signaling mechanism of the signaling probe (SP) shifting different from the classical conformation/flexibility change mode endows the sensor with high sensitivity. Through using thiolated or no thiolated capturing probe (CP), two 3-probe sensing structures, sensor-1 and sensor-2, were designed and constructed. The systematical comparing research results show that both sensors exhibit some similarities or big differences in sensing performance. On the one hand, the similarity in structures determines the similarity in some aspects of signaling mechanism, background signal, signal changing form, anti-fouling ability and versatility; on the other hand, the slight difference in structures also results in two opposite hybridization modes of gradual increasing resistance and gradual decreasing resistance which can affect the hybridization efficiency between the assistant probe (AP) and the SP, further producing some big differences in sensing performance, for example, apparently different signal enhancement (SE) change, point mutation discrimination ability and response speed. Under the optimized fabrication and detection conditions, both sensors feature high sensitivity for target DNAs with the detection limits of ∼10 fM for sensor-1 and ∼7 fM for sensor-2, respectively. Among many acquired sensing virtues, the sensor-1 shows a peculiar specificity adjustability which is also a highlight in this work.

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

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

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

  13. Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis.

    Directory of Open Access Journals (Sweden)

    Irvan Luhung

    Full Text Available As bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended.This study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1 enhanced high temperature sonication during DNA extraction; 2 effect of sampling duration on DNA recoverability; and 3 an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement and quantitative polymerase chain reaction (qPCR.The findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%.

  14. Electronic transport through dsDNA based junction: a Fibonacci model

    Directory of Open Access Journals (Sweden)

    S A Ketabi

    2014-12-01

    Full Text Available A numerical study is presented to investigate the electronic transport properties through a synthetic DNA molecule based on a quasiperiodic arrangement of its constituent nucleotides. Using a generalized Green's function technique, the electronic conduction through the poly(GACT-poly(CTGA DNA molecule in a metal/DNA/metal model structure has been studied. Making use of a renormalization scheme we transform the Hamiltonian of double-stranded DNA (dsDNA molecule to an effective Hamiltonian corresponding to a one-dimensional chain in which the effective on-site energies are arranged as a quasiperiodic lattice according to Fibonacci sequence. The room temperature current-voltage characteristic of dsDNA has been investigated in this Fibonacci model and compared with those corresponding to poly(GACT-poly(CTGA DNA molecule. Our results indicate the main effect of the quasiperiodic arrangement of the nucleotides as the Fibonacci sequence on the electronic spectrum structure of the dsDNA is that the energy band gaps of the molecule have a tendency for suppression. The room temperature I-V characteristic of the DNA Fibonacci model shows a linear and ohmic-like behavior

  15. DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool

    Directory of Open Access Journals (Sweden)

    Jin-Young Park

    2009-11-01

    Full Text Available Recent advances in label free DNA hybridization sensors employing electrochemical impedance spectroscopy (EIS as a detection tool are reviewed. These sensors are based on the modulation of the blocking ability of an electrode modified with a probe DNA by an analyte, i.e., target DNA. The probe DNA is immobilized on a self-assembled monolayer, a conducting polymer film, or a layer of nanostructures on the electrode such that desired probe DNA would selectively hybridize with target DNA. The rate of charge transfer from the electrode thus modified to a redox indicator, e.g., [Fe(CN6]3–/4–, which is measured by EIS in the form of charge transfer resistance (Rct, is modulated by whether or not, as well as how much, the intended target DNA is selectively hybridized. Efforts made to enhance the selectivity as well as the sensitivity of DNA sensors and to reduce the EIS measurement time are briefly described along with brief future perspectives in developing DNA sensors.

  16. DNA Hybridization Sensors Based on Electrochemical Impedance Spectroscopy as a Detection Tool

    Science.gov (United States)

    Park, Jin-Young; Park, Su-Moon

    2009-01-01

    Recent advances in label free DNA hybridization sensors employing electrochemical impedance spectroscopy (EIS) as a detection tool are reviewed. These sensors are based on the modulation of the blocking ability of an electrode modified with a probe DNA by an analyte, i.e., target DNA. The probe DNA is immobilized on a self-assembled monolayer, a conducting polymer film, or a layer of nanostructures on the electrode such that desired probe DNA would selectively hybridize with target DNA. The rate of charge transfer from the electrode thus modified to a redox indicator, e.g., [Fe(CN)6]3−/4−, which is measured by EIS in the form of charge transfer resistance (Rct), is modulated by whether or not, as well as how much, the intended target DNA is selectively hybridized. Efforts made to enhance the selectivity as well as the sensitivity of DNA sensors and to reduce the EIS measurement time are briefly described along with brief future perspectives in developing DNA sensors. PMID:22303136

  17. From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments

    Science.gov (United States)

    Recent technological advances have driven rapid development of DNA-based methods designed to facilitate detection and monitoring of invasive species in aquatic environments. These tools promise to significantly alleviate difficulties associated with traditional monitoring approac...

  18. P element excision in drosophila melanogaster and related drosophilids

    Science.gov (United States)

    The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlik...

  19. Effects of burn wound excision on bacterial colonization and invasion

    NARCIS (Netherlands)

    Barret, JP; Herndon, DN

    2003-01-01

    Rates of survival after thermal injury have improved in the past two decades, and rates of wound infections and sepsis have decreased during the same period. Early excision has been advocated as one of the major factors, but its safety and efficacy and the exact timing of burn excision are still und

  20. Adaptive Signal Processing Testbed signal excision software: User's manual

    Science.gov (United States)

    Parliament, Hugh A.

    1992-05-01

    The Adaptive Signal Processing Testbed (ASPT) signal excision software is a set of programs that provide real-time processing functions for the excision of interfering tones from a live spread-spectrum signal as well as off-line functions for the analysis of the effectiveness of the excision technique. The processing functions provided by the ASPT signal excision software are real-time adaptive filtering of live data, storage to disk, and file sorting and conversion. The main off-line analysis function is bit error determination. The purpose of the software is to measure the effectiveness of an adaptive filtering algorithm to suppress interfering or jamming signals in a spread spectrum signal environment. A user manual for the software is provided, containing information on the different software components available to perform signal excision experiments: the real-time excision software, excision host program, file processing utilities, and despreading and bit error rate determination software. In addition, information is presented describing the excision algorithm implemented, the real-time processing framework, the steps required to add algorithms to the system, the processing functions used in despreading, and description of command sequences for post-run analysis of the data.

  1. An Affinity Propagation-Based DNA Motif Discovery Algorithm

    Directory of Open Access Journals (Sweden)

    Chunxiao Sun

    2015-01-01

    Full Text Available The planted (l,d motif search (PMS is one of the fundamental problems in bioinformatics, which plays an important role in locating transcription factor binding sites (TFBSs in DNA sequences. Nowadays, identifying weak motifs and reducing the effect of local optimum are still important but challenging tasks for motif discovery. To solve the tasks, we propose a new algorithm, APMotif, which first applies the Affinity Propagation (AP clustering in DNA sequences to produce informative and good candidate motifs and then employs Expectation Maximization (EM refinement to obtain the optimal motifs from the candidate motifs. Experimental results both on simulated data sets and real biological data sets show that APMotif usually outperforms four other widely used algorithms in terms of high prediction accuracy.

  2. An Affinity Propagation-Based DNA Motif Discovery Algorithm.

    Science.gov (United States)

    Sun, Chunxiao; Huo, Hongwei; Yu, Qiang; Guo, Haitao; Sun, Zhigang

    2015-01-01

    The planted (l, d) motif search (PMS) is one of the fundamental problems in bioinformatics, which plays an important role in locating transcription factor binding sites (TFBSs) in DNA sequences. Nowadays, identifying weak motifs and reducing the effect of local optimum are still important but challenging tasks for motif discovery. To solve the tasks, we propose a new algorithm, APMotif, which first applies the Affinity Propagation (AP) clustering in DNA sequences to produce informative and good candidate motifs and then employs Expectation Maximization (EM) refinement to obtain the optimal motifs from the candidate motifs. Experimental results both on simulated data sets and real biological data sets show that APMotif usually outperforms four other widely used algorithms in terms of high prediction accuracy.

  3. Interference Excision in Spread Spectrum Communications Using Adaptive Positive Time-Frequency Analysis

    Directory of Open Access Journals (Sweden)

    Krishnan Sridhar

    2007-01-01

    Full Text Available This paper introduces a novel algorithm to excise single and multicomponent chirp-like interferences in direct sequence spread spectrum (DSSS communications. The excision algorithm consists of two stages: adaptive signal decomposition stage and directional element detection stage based on the Hough-Radon transform (HRT. Initially, the received spread spectrum signal is decomposed into its time-frequency (TF functions using an adaptive signal decomposition algorithm, and the resulting TF functions are mapped onto the TF plane. We then use a line detection algorithm based on the HRT that operates on the image of the TF plane and detects energy varying directional elements that satisfy a parametric constraint. Interference is modeled by reconstructing the corresponding TF functions detected by the HRT, and subtracted from the received signal. The proposed technique has two main advantages: (i it localizes the interferences on the TF plane with no cross-terms, thus facilitating simple filtering techniques based on thresholding of the TF functions, and is an efficient way to excise the interference; (ii it can be used for the detection of any directional interferences that can be parameterized. Simulation results with synthetic models have shown successful performance with linear and quadratic chirp interferences for single and multicomponent interference cases. The proposed method excises the interference even under very low SNR conditions of  dB, and the technique could be easily extended to any interferences that could be represented by a parametric equation in the TF plane.

  4. Measurement of oxidized and methylated DNA bases by HPLC with electrochemical detection.

    Science.gov (United States)

    Kaur, H; Halliwell, B

    1996-08-15

    Oxidative DNA damage is thought to be an important contributor to cancer development and to be affected by dietary constituents, so its accurate measurement is important. DNA methylation is recognized as an important mechanism affecting gene expression. In the present paper we describe an HPLC-with-electrochemical-detection procedure that allows rapid and sensitive measurement of four oxidized (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 8-hydroxyguanine, 8-hydroxyadenine) and three methylated (7-methylguanine, 1-methylguanine, O6-methylguanine) bases in acid hydrolysates of DNA. Guanine was also detected, but was clearly separated from the other bases.

  5. Platinum drugs and DNA repair mechanisms in lung cancer.

    Science.gov (United States)

    Bonanno, Laura; Favaretto, Adolfo; Rosell, Rafael

    2014-01-01

    The standard first-line treatment for around 80% of newly-diagnosed advanced non-small cell lung cancer (NSCLC) is chemotherapy. Currently, patients are allocated to chemotherapy on the basis of clinical conditions, comorbidities and histology. If feasible, platinum-based chemotherapy is considered as the most efficacious option. Due to the heterogeneity in terms of platinum-sensitivity among patients with NSCLC, great efforts have been made in order to identify molecular predictive markers of platinum resistance. Based on the mechanism of action of platinum, several components of DNA repair pathways have been investigated as potential predictive markers. The main DNA repair pathways involved in the repair of platinum-induced DNA damage are nucleotide excision repair and homologous recombination. The most studied potential predictive markers of platinum-sensitivity are Excision Repair Cross Complementing-1 (ERCC1) and Brest Cancer Type-I Susceptibility protein (BRCA1); however, increasing biological knowledge about DNA repair pathways suggests the potential clinical usefulness of integrated analysis of multiple DNA repair components.

  6. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    Science.gov (United States)

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

  7. Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Chikako Kiyohara, Kouichi Yoshimasu

    2007-01-01

    Full Text Available Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA, excision repair cross complementing group 1 (ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR = 1.30, 95% confidence interval (CI = 1.14 - 1.49. We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95. Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.

  8. Optimal Design of TS Fuzzy Control System Based on DNA Evolutionary Algorithm%采用DNA进化算法优化设计TS模糊控制器

    Institute of Scientific and Technical Information of China (English)

    翁妙凤

    2003-01-01

    The DNA evolutionary algorithm(DNA-EA)and the DNA genetic algorithm(DNA-GA)based on a new DNA encoding method are propsed based on the structure and the genetic mechanism of biological DNA. The DNA-EA and the DNA-GA are applied into the optimal design of TS fuzzy control system. The simulation results show the effectiveness of the two DNA algorithms, excellent self-learning capability. However, the DNA-EA is superior to the DNA-GA in the simulation performance.

  9. Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; Lin Yang; Jian-Tao Cui; Wen-Mei Li; Rui-Fang Guo; You-Yong Lu

    2002-01-01

    AIM: To elucidate molecular mechanism of chemopreventiveefficacies of garlic against human gastric cancer (HGC):METHODS: HGC cell line BGC823 was treated with Allitridi (akind of garlic extract) and Allitridi-treated and parentalBGC823 cDNA librarles were constructed respectively byusing λZAP Ⅱ vector. cDNA Representatinal DifferenceAnalysis (cDNA RDA) was perfonmed using BamH Ⅰ cutting-site and abundant ~DNA messages provided by the Iibrarles.Northern blot analysls was applied to identifythe obtaineddifference prnducts.RESULTS: Two specific cDNA fragments were obtained andcharacterized to be derived from homo sapiens folatereceptorα (FRα) gene and calcyclin gene respectively.Northern blot results showed a 4-fold increase in FRα geneexpression level and 9-fold increase in calcyclin mRNA levelin BGC823 cells after Allilridi treatment for 72 h.CONCLUSION: The method of cDNA RDA based on cDNAlibraries combines the high specificity of cDNA RDA withabundant cDNA messages in cDNA library; this expands theapplication of cDNA library and increases the specificity ofcDNA RDA. Up-regulstion of FRα gene and calcyclin geneexpressions induced by Allitridi provide valuable molecularevidence for theefficacy of garlic in treating HGC as well asother diseases.

  10. Local compression properties of double-stranded DNA based on a dynamic simulation

    CERN Document Server

    Lei, Xiaoling; Fang, Haiping

    2013-01-01

    The local mechanical properties of DNA are believed to play an important role in their biological functions and DNA-based nanomechanical devices. Using a simple sphere-tip compression system, the local radial mechanical properties of DNA are systematically studied by changing the tip size. The compression simulation results for the 16 nm diameter sphere tip are well consistent with the experimental results. With the diameter of the tip decreasing, the radial compressive elastic properties under external loads become sensitive to the tip size and the local DNA conformation. There appears a suddenly force break in the compression-force curve when the sphere size is less than or equal to 12 nm diameter. The analysis of the hydrogen bonds and base stacking interaction shows there is a local unwinding process occurs. During the local unwinding process, first the hydrogen bonds between complement base pairs are broken. With the compression aggregating, the local backbones in the compression center are unwound from ...

  11. A Real-Time de novo DNA Sequencing Assembly Platform Based on an FPGA Implementation.

    Science.gov (United States)

    Hu, Yuanqi; Georgiou, Pantelis

    2016-01-01

    This paper presents an FPGA based DNA comparison platform which can be run concurrently with the sensing phase of DNA sequencing and shortens the overall time needed for de novo DNA assembly. A hybrid overlap searching algorithm is applied which is scalable and can deal with incremental detection of new bases. To handle the incomplete data set which gradually increases during sequencing time, all-against-all comparisons are broken down into successive window-against-window comparison phases and executed using a novel dynamic suffix comparison algorithm combined with a partitioned dynamic programming method. The complete system has been designed to facilitate parallel processing in hardware, which allows real-time comparison and full scalability as well as a decrease in the number of computations required. A base pair comparison rate of 51.2 G/s is achieved when implemented on an FPGA with successful DNA comparison when using data sets from real genomes.

  12. High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on DNA from T7 bacteriophage and two novel forms of T4 bacteriophage.

    Science.gov (United States)

    Clark, R W; Wever, G H; Wiberg, J S

    1980-01-01

    The DNA molecules from T7 bacteriophage and a recently obtained mutant form of T4D were studied. The DNA of this T4 mutant contains cytosine in place of all of the glucosylated hydroxymethylcytosines normally present in T4. Molecular weights were measured with an electron microscope technique, and sedimentation coefficients were determined in isokinetic sucrose gradients. T7 DNA was found to have an Mr of 26.5 x 10(6). The T4 mutant, which we have termed T4c, produces two distinct phage head and DNA size clases. DNA from the standard heads (T4c DNA) has an Mr of 114.9 x 10(6), and DNA from the petite heads (T4cp DNA) has an Mr of 82.9 x 10(6). This enabled the derivation of an equation of sedimentation coefficient at zero concentration corrected to water at 20 degrees C versus Mr for the molecular weight range of 25 x 10(6) to 115 x 10(6) that is based solely on cytosine-containing DNA standards, thereby avoiding possible anomalies introduced by the glucosylation and hydroxymethylation of cytosine. The theory of Gray et al. provided the best description of the sedimentation coefficient versus Mr relationship, based on the sedimentation coefficients and the molecular weights of the three DNA standards and other evidence.

  13. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod-odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover,the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  14. Pros and cons of methylation-based enrichment methods for ancient DNA

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio;

    2015-01-01

    The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules...... containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD...... enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved...

  15. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  16. A novel single-stranded DNA detection method based on organic semiconductor heterojunction

    Science.gov (United States)

    Gu, Wen; Liu, Hongbo; Zhang, Xia; Zhang, Hao; Chen, Xiong; Wang, Jun

    2016-12-01

    We demonstrate a novel DNA detection method with low-cost and disposable advantages by utilizing F16CuPc/CuPc planar organic heterojunction device. Single-stranded DNA (ssDNA) molecules have been well immobilized on the surface of CuPc film observed by atomic force microscopy, producing an obvious electrical response of the device. The conductivity of the organic heterojunction film was significantly increased by ssDNA immobilization because ssDNA molecules brought additional positive charges at heterojunction interface. Furthermore, the thickness dependence of CuPc upper layer on the electrical response was studied to optimize the sensitivity. This study will be helpful for the development of organic heterojunction based biosensors.

  17. DNA Isolation and GC Base Composition of Four Root-knot Nematode (Meloidogyne spp.) Genomes.

    Science.gov (United States)

    Pableo, E C; Triantaphyllou, A C; Kloos, W E

    1988-01-01

    Phenol extraction and cesium trifluoroacetate ultracentrifugation were compared for efficiency in the extraction of DNA from eggs and second-stage juveniles of four species of Meloidogyne. The second method proved to be more satisfactory in that it yielded larger amounts of DNA, shortened the extraction period, and reduced sample handling by eliminating phenol and ether extraction and RNAse treatment. It also made possible the extraction of DNA: from more than one sample at a time. The mean base compositions (% GC) of the total DNA of M. incognita, M. javanica, M. arenaria, and M. hapla, as determined by thermal denaturation tests, were quite similar, as they ranged only between 31 and 33%. Similarly, the thermal stability of the DNA of all four species covered a narrow range from 82.97 to 83.63 C.

  18. 顺势疗法药物山金车30C通过上调核苷酸切除修复基因的表达减少紫外线照射后大肠杆菌的DNA损伤%Potential of the homeopathic remedy, Arnica Montana 30C,to reduce DNA damage in Escherichia coli exposed to ultraviolet irradiation through up-regulation of nucleotide excision repair genes

    Institute of Scientific and Technical Information of China (English)

    Sreemanti Das; Santu Kumar Saha; Arnab De; Durba Das; Anisur Rahman Khuda-Bukhs

    2012-01-01

    目的:检测高度稀释的顺势疗法药物山金车30C是否能够调节暴露于紫外线照射下的大肠杆菌的核苷酸切除修复基因的表达.方法:大肠杆菌在标准培养基中培养至对数阶段,然后接受亚致死剂量的紫外线照射(25和50 J/m2分别照射22.5和45 s).接受不同剂量紫外线照射的大肠杆菌分别与山金车30C及安慰剂30C共同培养,90 min后检测其DNA损伤情况及氧化应激状态.采用多种方法及指标如彗星实验、梯度凝胶电泳、细胞内活性氧生成及测量其他生物活性指标如过氧化物歧化酶、过氧化氢酶及谷胱甘肽衡量DNA损伤情况及细胞氧化应激状态.逆转录聚合酶链反应检测大肠杆菌细胞紫外线损伤修复基因uvrA、B、C(核苷酸切除修复基因)mRNA的表达情况.结果:接受照射后的大肠杆菌出现了DNA损伤及氧化应激反应,表现为细胞内活性氧生成增加及过氧化物歧化酶、过氧化氢酶和谷胱甘肽活性降低.与安慰剂组相比,山金车30C降低了大肠杆菌的DNA损伤及氧化应激反应,表现为细胞内活性氧生成减少及过氧化物歧化酶、过氧化氢酶和谷胱甘肽活性增强.与对照组相比,山金车30C上调了大肠杆菌细胞紫外线损伤修复基因的表达.结论:山金车30C能够通过上调紫外线损伤修复基因的表达修复紫外线引起的大肠杆菌细胞的DNA损伤,并通过减少细胞内活性氧的生成及调节抗氧化酶活性降低细胞的氧化应激反应.%OBJECTIVE:To examine to what degree an ultra-highly diluted homeopathic remedy,Arnica Montana 30C (AM-30C),used in the treatment of shock and injury,can modulate the expression of nucleotide excision repair genes in Escherichia coii exposed to ultraviolet (UV) irradiation.METHODS:E.coli were cultured to their log phase in a standard Luria-Bertani medium and then exposed to sublethal doses of UV irradiation at 25 and 50 J/m2 for 22.5 and 45 s,respectively.The UV

  19. Helicobacter pylori Infection Induces Genetic Instability of Nuclear and Mitochondrial DNA in Gastric Cells

    DEFF Research Database (Denmark)

    Machado, Ana Manuel; Figueiredo, Ceu; Touati, Eliette;

    2009-01-01

    Purpose: Helicobacter pylori is a major cause of gastric carcinoma. To investigate a possible link between bacterial infection and genetic instability of the host genome, we examined the effect of H. pylori infection on known cellular repair pathways in vitro and in vivo. Moreover, various types...... of genetic instabilities in the nuclear and mitochondrial DNA (mtDNA) were examined. Experimental Design: We observed the effects of H pylori infection on a gastric cell line (AGS), on C57BL/6 mice, and on individuals with chronic gastritis. In AGS cells, the effect of H pylori infection on base excision...... cells and chronic gastritis tissue were determined by PCR, single-stranded conformation polymorphism, and sequencing. H pylori vacA and cagA genotyping was determined by multiplex PCR and reverse hybridization. Results: Following H pylori infection, the activity and expression of base excision repair...

  20. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    Science.gov (United States)

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method.

  1. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    Energy Technology Data Exchange (ETDEWEB)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae.

  2. Lingual Thyroid Excision with Transoral Robotic Surgery

    Directory of Open Access Journals (Sweden)

    Elif Ersoy Callıoglu

    2015-01-01

    Full Text Available Ectopic thyroid gland may be detected at any place between foramen caecaum and normal thyroid localization due to inadequacy of the embryological migration of the thyroid gland. It has a prevalence varying between 1/10.000 and 1/100000 in the community. Usually follow-up without treatment is preferred except for obstructive symptoms, bleeding, and suspicion of malignity. Main symptoms are dysphagia, dysphonia, bleeding, dyspnea, and obstructive sleep apnea. In symptomatic cases, the first described method in surgical treatment is open approach since it is a region difficult to have access to. However, this approach has an increased risk of morbidity and postoperative complications. Transoral robotic surgery, which is a minimally invasive surgical procedure, has advantages such as larger three-dimensional point of view and ease of manipulation due to robotic instruments. In this report, a case at the age of 49 who presented to our clinic with obstructive symptoms increasing within the last year and was found to have lingual thyroid and underwent excision of ectopic thyroid tissue by da Vinci surgical system is presented.

  3. Complete mesocolic excision: Techniques and outcomes

    Institute of Scientific and Technical Information of China (English)

    Nikoletta; Dimitriou; John; Griniatsos

    2015-01-01

    Complete mesocolic excision(CME) for the treatment of colon cancer was first introduced in the West in 2008. The first aim of this procedure is to remove the afflicted colon and its accessory lymphovascular supply by resecting the colon and mesocolon in an intact envelope of visceral peritoneum, which holds potentiallyinvolved lymph nodes. The second component of CME is a central vascular tie to remove completely all lymph nodes in the central(vertical) direction. In its original iteration, CME was performed via laparotomy, although many centers preferentially perform laparoscopic surgery, with its associated benefits and similar oncolo-gical outcomes, as the standard treatment for colonic cancer. Here, we present the surgical techniques for CME in open and laparoscopic surgery, as well as the surgical, pathological and oncological outcomes of the procedure that are available to date. Because there are no randomized control trials comparing CME to "standard" colon surgery, the principles underlying CME seem anatomical and logical, and the results published from the Far East, reporting an 80% 5-year survival rate for Stage III cancer, should guide us.

  4. Surface ligation-based resonance light scattering analysis of methylated genomic DNA on a microarray platform.

    Science.gov (United States)

    Ma, Lan; Lei, Zhen; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-05-10

    DNA methylation is a crucial epigenetic modification and is closely related to tumorigenesis. Herein, a surface ligation-based high throughput method combined with bisulfite treatment is developed for analysis of methylated genomic DNA. In this method, a DNA microarray is employed as a reaction platform, and resonance light scattering (RLS) of nanoparticles is used as the detection principle. The specificity stems from allele-specific ligation of Taq DNA ligase, which is further enhanced by improving the fidelity of Taq DNA ligase in a heterogeneous reaction. Two amplification techniques, rolling circle amplification (RCA) and silver enhancement, are employed after the ligation reaction and a gold nanoparticle (GNP) labeling procedure is used to amplify the signal. As little as 0.01% methylated DNA (i.e. 2 pmol L(-1)) can be distinguished from the cocktail of methylated and unmethylated DNA by the proposed method. More importantly, this method shows good accuracy and sensitivity in profiling the methylation level of genomic DNA of three selected colonic cancer cell lines. This strategy provides a high throughput alternative with reasonable sensitivity and resolution for cancer study and diagnosis.

  5. Anhydrous crystals of DNA bases are wide gap semiconductors.

    Science.gov (United States)

    Maia, F F; Freire, V N; Caetano, E W S; Azevedo, D L; Sales, F A M; Albuquerque, E L

    2011-05-07

    We present the structural, electronic, and optical properties of anhydrous crystals of DNA nucleobases (guanine, adenine, cytosine, and thymine) found after DFT (Density Functional Theory) calculations within the local density approximation, as well as experimental measurements of optical absorption for powders of these crystals. Guanine and cytosine (adenine and thymine) anhydrous crystals are predicted from the DFT simulations to be direct (indirect) band gap semiconductors, with values 2.68 eV and 3.30 eV (2.83 eV and 3.22 eV), respectively, while the experimentally estimated band gaps we have measured are 3.83 eV and 3.84 eV (3.89 eV and 4.07 eV), in the same order. The electronic effective masses we have obtained at band extremes show that, at low temperatures, these crystals behave like wide gap semiconductors for electrons moving along the nucleobases stacking direction, while the hole transport are somewhat limited. Lastly, the calculated electronic dielectric functions of DNA nucleobases crystals in the parallel and perpendicular directions to the stacking planes exhibit a high degree of anisotropy (except cytosine), in agreement with published experimental results.

  6. Triazene-Based Traceless Linkers for DNA-Directed Chemistry and Development of Methods for Linking Nanomaterials to DNA Origami

    DEFF Research Database (Denmark)

    Hejesen, Christian

    2013-01-01

    Denne ph.d.-afhandling indeholder fire kapitler, der hver beskriver forskellige områder indenfor det videnskabelig felt: DNA nanoteknologi. I kapitel 1 bliver der givet en generel introduktion til DNA og DNA-dirigeret kemi. Først bliver historien bag, og opdagelsen af DNA beskrevet, efterfulgt af...

  7. Image Encryption Algorithm Based on Dynamic DNA Coding and Chen’s Hyperchaotic System

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2016-01-01

    Full Text Available With the development of national information processes, specific image information from secret departments or individuals is often required to be confidentially transmitted. Numerous image encryption methods exist, especially since the initial value sensitivity and other characteristics of chaos theory and chaos theory-based encryption have become increasingly important in recent years. At present, DNA coding constitutes a new research direction of image encryption that uses the four base pairs of DNA code and image pixel values to establish a special correspondence, in order to achieve pixel diffusion. There are eight DNA encoding rules, and current methods of selecting the DNA encoding rules are largely fixed. Thus, the security of encoded data is not high. In this paper, we use the Lorenz chaotic system, Chen’s hyperchaotic system, and the DNA encoding combination and present a new image encryption algorithm that can dynamically select eight types of DNA encoding rules and eight types of DNA addition and subtraction rules, with significant improvements in security. Through simulation experiments and histograms, correlations, and NPCR analyses, we have determined that the algorithm possesses numerous desirable features, including good encryption effects and antishear and antinoise performances.

  8. Label-Free Detection of Ag+ Based on Gold Nanoparticles and Ag+-Specific DNA.

    Science.gov (United States)

    Pu, Wendan; Zhao, Zhao; Wu, Liping; Liu, Yue; Zhao, Huawen

    2015-08-01

    A sensitive label-free method was presented for the determination of silver ion (Ag+) in this paper. Cytosine-rich DNA (C-DNA) was used as Ag+ specific DNA. Without Ag+ in the solution, fluorescence of fluorescein (FAM) is quenched by C-DNA stabilized gold nanoparticles (AuNPs) in high salt environment. When Ag+ is present in the solution, however, Ag+-mediated cytosine-Ag+-cytosine (C-Ag+-C) base pairs induced the C-DNA folding into a hairpin structure, which can not stabilize AuNPs in high salt environment, thus causing AuNPs aggregation. After centrifugation to remove the aggregated AuNPs, the quenching ability of the supernatant for FAM is decreased and the fluorescence intensity of solution increases with increasing the Ag+ concentration. Due to the highly specific interaction of the C-DNA towards Ag+ and the strong fluorescent quenching ability of AuNPs for FAM, the method has high selectivity and sensitivity for Ag+. Under the optimal conditions, the fluorescence intensity at 515 nm increased linearly with the concentration of Ag+ ranging from 15 nM to 700 nM, and the detection limit was determined as 6 nM based on 3 σ/slope. This method is simple, sensitive, and may be applied to other detection systems by selecting the appropriate DNA sequences.

  9. An algorithm for the study of DNA sequence evolution based on the genetic code.

    Science.gov (United States)

    Sirakoulis, G Ch; Karafyllidis, I; Sandaltzopoulos, R; Tsalides, Ph; Thanailakis, A

    2004-11-01

    Recent studies of the quantum-mechanical processes in the DNA molecule have seriously challenged the principle that mutations occur randomly. The proton tunneling mechanism causes tautomeric transitions in base pairs resulting in mutations during DNA replication. The meticulous study of the quantum-mechanical phenomena in DNA may reveal that the process of mutagenesis is not completely random. We are still far away from a complete quantum-mechanical model of DNA sequence mutagenesis because of the complexity of the processes and the complex three-dimensional structure of the molecule. In this paper we have developed a quantum-mechanical description of DNA evolution and, following its outline, we have constructed a classical model for DNA evolution assuming that some aspects of the quantum-mechanical processes have influenced the determination of the genetic code. Conversely, our model assumes that the genetic code provides information about the quantum-mechanical mechanisms of mutagenesis, as the current code is the product of an evolutionary process that tries to minimize the spurious consequences of mutagenesis. Based on this model we develop an algorithm that can be used to study the accumulation of mutations in a DNA sequence. The algorithm has a user-friendly interface and the user can change key parameters in order to study relevant hypotheses.

  10. A DNA methylation-based definition of biologically distinct breast cancer subtypes.

    Science.gov (United States)

    Stefansson, Olafur A; Moran, Sebastian; Gomez, Antonio; Sayols, Sergi; Arribas-Jorba, Carlos; Sandoval, Juan; Hilmarsdottir, Holmfridur; Olafsdottir, Elinborg; Tryggvadottir, Laufey; Jonasson, Jon G; Eyfjord, Jorunn; Esteller, Manel

    2015-03-01

    In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation-based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype-specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal-B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal-like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation-based subtypes, Epi-LumB and Epi-Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation-based subtypes thereby providing valuable information on disease prognosis.

  11. TAA Polyepitope DNA-Based Vaccines: A Potential Tool for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Roberto Bei

    2010-01-01

    Full Text Available DNA-based cancer vaccines represent an attractive strategy for inducing immunity to tumor associated antigens (TAAs in cancer patients. The demonstration that the delivery of a recombinant plasmid encoding epitopes can lead to epitope production, processing, and presentation to CD8+ T-lymphocytes, and the advantage of using a single DNA construct encoding multiple epitopes of one or more TAAs to elicit a broad spectrum of cytotoxic T-lymphocytes has encouraged the development of a variety of strategies aimed at increasing immunogenicity of TAA polyepitope DNA-based vaccines. The polyepitope DNA-based cancer vaccine approach can (a circumvent the variability of peptide presentation by tumor cells, (b allow the introduction in the plasmid construct of multiple immunogenic epitopes including heteroclitic epitope versions, and (c permit to enroll patients with different major histocompatibility complex (MHC haplotypes. This review will discuss the rationale for using the TAA polyepitope DNA-based vaccination strategy and recent results corroborating the usefulness of DNA encoding polyepitope vaccines as a potential tool for cancer therapy.

  12. Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Eiko E Kuramae

    Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

  13. Wound Complications and Perineal Pain After Extralevator Versus Standard Abdominoperineal Excision

    DEFF Research Database (Denmark)

    Colov, Emilie P; Klein, Mads; Gögenur, Ismail

    2016-01-01

    BACKGROUND: Extralevator abdominoperineal excision was introduced as an alternative to conventional abdominoperineal excision for low rectal cancers. The perineal dissection is more extensive with extralevator abdominoperineal excision and leaves a greater defect. OBJECTIVE: The aim of this study...

  14. 77 FR 43157 - Disregarded Entities and the Indoor Tanning Services Excise Tax; Correction

    Science.gov (United States)

    2012-07-24

    ... Excise Tax; Correction AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Correcting amendment... qualified subchapter S subsidiaries) and the indoor tanning services excise tax. DATES: This correction is... Employment taxes, Estate taxes, Excise taxes, Gift taxes, Income taxes, Penalties, Reporting...

  15. 77 FR 37806 - Disregarded Entities and the Indoor Tanning Services Excise Tax

    Science.gov (United States)

    2012-06-25

    ... Services Excise Tax AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Final and temporary... (including qualified subchapter S subsidiaries) and the indoor tanning services excise tax. These regulations affect disregarded entities responsible for collecting the indoor tanning services excise tax and...

  16. Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

    Science.gov (United States)

    Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

    2015-10-01

    DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR

  17. DNA repair: Dynamic defenders against cancer and aging

    Energy Technology Data Exchange (ETDEWEB)

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

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

  18. Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test; Phenotypage de la reparation de l'ADN de lignees Xeroderma pigmentosum, par un test in vitro multiparametrique

    Energy Technology Data Exchange (ETDEWEB)

    Raffin, A.L.

    2009-06-15

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

  19. [The application of mitochondrial genomics to forensic investigations based on human mitochondrial DNA testing].

    Science.gov (United States)

    Skonieczna, Katarzyna; Bednarek, Jarosław; Rogalla, Urszula; Woźniak, Marcin; Gorzkiewicz, Marta; Linkowska, Katarzyna; Duleba, Anna; Sliwka, Karol; Grzybowski, Tomasz

    2012-01-01

    In this study we present two forensic cases where mitochondrial DNA HVS I and HVS II haplotypes of evidentiary hairs match reference samples. Based on the information retrieved from mtDNA coding region of reference material, we selected single nucleotide polymorphisms (SNPs) located outside the HVS I and HVS II regions that could increase the informativeness of mtDNA analysis. The SNPs were typed via SNaPshot or dideoxy sequencing technology. In both cases the SNP results allowed for unambiguous exlusion of the evidence and for determining that reference samples originated from the same person.

  20. Role of dissolved salts in thermophoresis of DNA: lattice-Boltzmann-based simulations.

    Science.gov (United States)

    Hammack, Audrey; Chen, Yeng-Long; Pearce, Jennifer Kreft

    2011-03-01

    We use a lattice Boltzmann based Brownian dynamics simulation to investigate the dependence of DNA thermophoresis on its interaction with dissolved salts. We find the thermal diffusion coefficient D{T} depends on the molecule size, in contrast with previous